Publications

Opsins are G protein-coupled receptors (GPCRs) that have evolved to detect light stimuli and initiate intracellular signaling cascades. Their role as signal transducers is critical to light perception across the animal kingdom. Opsins covalently bind to the chromophore 11-cis retinal, which isomerizes to the all-trans isomer upon photon absorption, causing conformational changes that result in receptor activation. Monostable opsins, responsible for vision in vertebrates, release the chromophore after activation and must bind another retinal molecule to remain functional. In contrast, bistable opsins, responsible for non-visual light perception in vertebrates and for vision in invertebrates, absorb a second photon in the active state to return the chromophore and protein to the inactive state. Structures of bistable opsins in the activated state have proven elusive, limiting our understanding of how they function as bidirectional photoswitches. Here we present active state structures of a bistable opsin, jumping spider rhodopsin isoform-1 (JSR1), in complex with its downstream signaling partners, the G_i and G_q heterotrimers. These structures elucidate key differences in the activation mechanisms between monostable and bistable opsins, offering essential insights for the rational engineering of bistable opsins into diverse optogenetic tools to control G protein signaling pathways.

The fundamental question of \u201cwhat is the transport path of electrons through proteins?\u201d initially introduced while studying long-range electron transfer between localized redox centers in proteins in vivo is also highly relevant to the transport properties of solid-state, dry metalproteinmetal junctions. Here, we report conductance measurements of such junctions, Au-(Azurin monolayer ensemble)-Bismuth (Bi) ones, with well-defined nanopore geometry and ~10³ proteins/pore. Our results can be understood as follows. (1) Transport is via two interacting conducting channels, characterized by different spatial and time scales. The slow and spatially localized channel is associated with the Cu center of Azurin and the fast delocalized one with the proteins polypeptide matrix. Transport via the slow channel is by a sequential (noncoherent) process and in the second one by direct, off-resonant tunneling. (2) The two channels are capacitively coupled. Thus, with a change in charge occupation of the weakly coupled (metal center) channel, the broad energy level manifold, responsible for off-resonance tunneling, shifts, relative to the electrodes Fermi levels. In this process, the off-resonance (fast) channel dominates transport, and the slow (redox) channel, while contributing only negligibly directly, significantly affects transport by intramolecular gating.

In recent years it became apparent that, in mammals, rhodopsin and other opsins, known to act as photosensors in the visual system, are also present in spermatozoa, where they function as highly sensitive thermosensors for thermotaxis. The intriguing question how a well-conserved protein functions as a photosensor in one type of cells and as a thermosensor in another type of cells is unresolved. Since the moiety that confers photosensitivity on opsins is the chromophore retinal, we examined whether retinal is substituted in spermatozoa with a thermosensitive molecule. We found by both functional assays and mass spectrometry that retinal is present in spermatozoa and required for thermotaxis. Thus, starvation of mice for vitamin A (a precursor of retinal) resulted in loss of sperm thermotaxis, without affecting motility and the physiological state of the spermatozoa. Thermotaxis was restored after replenishment of vitamin A. Using reversed-phase ultra-performance liquid chromatography mass spectrometry, we detected the presence of retinal in extracts of mouse and human spermatozoa. By employing UltraPerformance convergence chromatography, we identified a unique retinal isomer in the sperm extractstri-cis retinal, different from the photosensitive 11-cis isomer in the visual system. The facts (a) that opsins are thermosensors for sperm thermotaxis, (b) that retinal is essential for thermotaxis, and (c) that tri-cis retinal isomer uniquely resides in spermatozoa and is relatively thermally unstable, suggest that tri-cis retinal is involved in the thermosensing activity of spermatozoa.

A key conundrum of biomolecular electronics is efficient electron transport (ETp) through solid-state junctions up to 10 nm, often without temperature activation. Such behavior challenges known charge transport mechanisms, especially via nonconjugated molecules such as proteins. Single-step, coherent quantum-mechanical tunneling proposed for ETp across small protein, 2-3 nm wide junctions, but it is problematic for larger proteins. Here we exploit the ability of bacteriorhodopsin (bR), a well-studied, 4-5 nm long membrane protein, to assemble into well-defined single and multiple bilayers, from ∼9 to 60 nm thick, to investigate ETp limits as a function of junction width. To ensure sufficient signal/noise, we use large area (∼10^-3 cm²) Au-protein-Si junctions. Photoemission spectra indicate a wide energy separation between electrode Fermi and the nearest protein-energy levels, as expected for a polymer of mostly saturated components. Junction currents decreased exponentially with increasing junction width, with uniquely low length-decay constants (0.05-0.5 nm^-1). Remarkably, even for the widest junctions, currents are nearly temperature-independent, completely so below 160 K. While, among other things, the lack of temperature-dependence excludes, hopping as a plausible mechanism, coherent quantum-mechanical tunneling over 60 nm is physically implausible. The results may be understood if ETp is limited by injection into one of the contacts, followed by more efficient charge propagation across the protein. Still, the electrostatics of the protein films further limit the number of charge carriers injected into the protein film. How electron transport across dozens of nanometers of protein layers is more efficient than injection defines a riddle, requiring further study.

Microbial rhodopsin (also called retinal protein)carotenoid conjugates represent a unique class of light-harvesting (LH) complexes, but their specific interactions and LH properties are not completely elucidated as only few rhodopsins are known to bind carotenoids. Here, we report a natural sodium-ion (Na+)-pumping Nonlabens (Donghaeana) dokdonensis rhodopsin (DDR2) binding with a carotenoid salinixanthin (Sal) to form a thermally stable rhodopsincarotenoid complex. Different spectroscopic studies were employed to monitor the retinalcarotenoid interaction as well as the thermal stability of the protein, while size-exclusion chromatography (SEC) and homology modeling are performed to understand the protein oligomerization process. In analogy with that of another Na+-pumping protein Krokinobacter eikastus rhodopsin 2 (KR2), we propose that DDR2 (studied concentration range: 2 × 106 to 4 × 105 M) remains mainly as a pentamer at room temperature and neutral pH, while heating above 55 °C partially converted it into a thermally less stable oligomeric form of the protein. This process is affected by both the pH and concentration. At high concentrations (4 × 105 to 2 × 104 M), the protein adopts a pentamer form reflected in the excitonic circular dichroism (CD) spectrum. In the presence of Sal, the thermal stability of DDR2 is increased significantly, and the pigment is stable even at 85 °C. The results presented could have implications in designing stable rhodopsincarotenoid antenna complexes.

We demonstrate that the direction of current rectification via one of natures most efficient light-harvesting systems, the photosystem 1 complex (PS1), can be controlled by its orientation on Au substrates. Molecular self-assembly of the PS1 complex using four different linkers with distinct functional head groups that interact by electrostatic and hydrogen bonds with different surface parts of the entire protein PS1 complex was used to tailor the PS1 orientation. We observe an orientation-dependent rectification in the currentvoltage characteristics for linker/PS1 molecule junctions. Results of an earlier study using a surface two-site PS1 mutant complex having its orientation set by covalent binding to the Au substrate supports our conclusion. Currentvoltagetemperature measurements on the linker/PS1 complex indicate off-resonant tunneling as the main electron transport mechanism. Our ultraviolet photoemission spectroscopy results highlight the importance of the protein orientation for the energy level alignment and provide insight into the charge transport mechanism via the PS1 transport chain.

The electron transport (ETp) efficiency of solid-state protein-mediated junctions is highly influenced by the presence of electron-rich organic cofactors or transition metal ions. Hence, we chose to investigate an interesting cofactor-free non-redox protein, streptavidin (STV), which has unmatched strong binding affinity for an organic small-molecule ligand, biotin, which lacks any electron-rich features. We describe for the first time meso-scale ETp via electrical junctions of STV monolayers and focus on the question of whether the rate of ETp across both native and thiolated STV monolayers is influenced by ligand binding, a process that we show to cause some structural conformation changes in the STV monolayers. Au nanowire-electrodeprotein monolayermicroelectrode junctions, fabricated by modifying an earlier procedure to improve the yields of usable junctions, were employed for ETp measurements. Our results on compactly integrated, dense, uniform, ∼3 nm thick STV monolayers indicate that, notwithstanding the slight structural changes in the STV monolayers upon biotin binding, there is no statistically significant conductance change between the free STV and that bound to biotin. The ETp temperature (T) dependence over the 80300 K range is very small but with an unusual, slightly negative (metallic-like) dependence toward room temperature. Such dependence can be accounted for by the reversible structural shrinkage of the STV at temperatures below 160 K.

Photoreceptor proteins play a critical role in light utilization for energy conversion and environmental sensing. Rhodopsin is a prototypical photoreceptor protein containing a retinal group that functions as a light-receptive site. It is essential to characterize the structure of the retinal chromophore because the chromophore structure, along with retinalprotein interactions, regulates which wavelengths of light are absorbed. Resonance Raman spectroscopy is a powerful tool to characterize chromophore structures in proteins. The resonance Raman spectra of heliorhodopsins, a recently discovered rhodopsin family, were previously reported to exhibit two intense ethylenic CC stretching bands never observed for type-1 rhodopsins. Here, we show that the double-band feature in the ethylenic CC stretching modes is not due to structural inhomogeneity but rather to the retinal polyene chains linear structure. It contrasts with bent all-trans chromophore in type-1 rhodopsins. The linear structure of the chromophore results from weak atomic contacts between the 13-methyl group and a nearby Trp side chain, which can slow thermal reisomerization in the photocycle. It is possible that the deceleration of reisomerization increases the lifetime of the signaling intermediate for photosensory function.

Immunoglobulin M (IgM) antibodies hold promise as anticancer drugs and as agents for promoting immune homeostasis. This promise has not been realized due to low expression levels in mammalian cells producing IgM class antibodies, and the failure of protein A chromatography for IgM purification. Here, we describe a nonchromatographic platform for quantitatively capturing IgMs at neutral pH, which is then recovered with 86%94% yield and >95% purity at pH 3. The platform contains micelles conjugated with the [(bathophenanthroline)3:Fe2+] amphiphilic complex. Inclusion of amino acid monomers, for example, phenylalanine or tyrosine, during conjugation of detergent micelles, allows subsequent extraction of IgMs at close to neutral pH. With the successful implementation of this purification platform for both polyclonal humans and bovine IgMs, we anticipate similar results for monoclonal IgMs, most relevant for the pharmaceutical industry.

In the decades'-long quest for high-quality membrane protein (MP) crystals, non-ionic detergent micelles have primarily served as a passive shield against protein aggregation in aqueous solution and/or as a conformation stabilizing environment. We have focused on exploiting the physical chemistry of detergent micelles in order to direct intrinsic MP/detergent complexes to assemble via conjugation under ambient conditions, thereby permitting finely tuned control over the micelle cloud point. In the current work, three commercially available amphiphilic, bipyridine chelators in combination with Fe
or Ni
were tested for their ability to conjugate non-ionic detergent micelles both in the presence and absence of an encapsulated bacteriorhodopsin molecule. Water-soluble chelators were added, and results were monitored with light microscopy and dynamic light scattering (DLS). [Bipyridine:metal] complexes produced micellar conjugates, which appeared as oil-rich globules (10-200 μm) under a light microscope. DLS analysis demonstrated that micellar conjugation is complete 20 min after the introduction of the amphiphilic complex, and that the conjugation process can be fully or partially reversed with water-soluble chelators. This process of controlled conjugation/deconjugation under nondenaturing conditions provides broader flexibility in the choice of detergent for intrinsic MP purification and conformational flexibility during the crystallization procedure.

To explain what drives us to study electron transport (ETp) through electrode/protein/electrode solid-state junctions (cf. Figure 1) we present some of the reasons, mostly in the form of the following questions:
1. Scientific curiosity: How can electron transport take place through nonconjugated, flexible, polyelectrolytic macromolecules? Answering this question is also driven by intense current interest to understand ETp via so-called bacterial nanowires. (1−3)
2. Biological implications and relevance:Can we learn from understanding ETp via proteins also about their role in biological electron transfer (ET)?
3. Physico-chemical insights: Which constituting elements and properties of proteins are involved in effective electron transport? The following can be singled out:
a. primary, secondary, and tertiary structure;
b. π-electron content and H-bonding character of amino-acid residues;
c. cofactors and their redox properties; alternatively, these can be described in terms of:
i. the (electronic) energy levels of a cofactors HOMO and LUMO;
ii. the energy difference between these levels, and between each of these levels and the electrode Fermi level; (51)
iii. the difference between the electrochemical potentials of the electrodes (= Fermi level) and of the protein (≈ redox potential (51)).
4. Potential applications: Can proteins serve as components of electronic devices as part of true bioelectronics?

Understanding the charge transport properties of proteins at the molecular scale is crucial for the development of novel bioelectronic devices. In this contribution, we report on the preparation and electrical characterization of thin films of bacteriorhodopsin grafted on the surface of titanium nitride via aminophosphonate linkers. Thickness analysis using atomic force microscopy revealed a protein film thickness of 8.2±1.5 nm, indicating the formation of a protein bilayer. Electrical measurements were carried out in the dry state, in a vertical arrangement with a eutectic gallium-indium (EGaIn) or an evaporated Ti/Au top contact. DC current-voltage measurements yielded comparable effective tunneling decay constants β∼0.13A-1 for the EGaIn top contact and ∼0.15A-1 for the Ti/Au top contact. The results presented herein may establish a novel platform for studying charge transport via protein molecules in a solid-state device configuration.

Heliorhodopsins are a recently discovered diverse retinal protein family with an inverted topology of the opsin where the retinal protonated Schiff base proton is facing the cell cytoplasmic side in contrast to type 1 rhodopsins. To explore whether light-induced retinal double-bond isomerization is a prerequisite for triggering protein conformational alterations, we utilized the retinal oxime formation reaction and thermal denaturation of a native heliorhodopsin of Thermoplasmatales archaeon SG8-52-1 (TaHeR) as well as a trans-locked retinal analogue (TaHeRL) in which the critical C13═C14 double-bond isomerization is prevented. We found that both reactions are light-accelerated not only in the native but also in the \u201clocked\u201d pigment despite lacking any isomerization. It is suggested that light-induced charge redistribution in the retinal excited state polarizes the protein and triggers protein conformational perturbations that thermally decay in microseconds. The extracted activation energy and the frequency factor for both the reactions reveal that the light enhancement of TaHeR differs distinctly from the earlier studied type 1 microbial rhodopsins.

A central issue in protein electronics is how far the structural stability of the protein is preserved under the very high electrical field that it will experience once a bias voltage is applied. This question is studied on the redox protein Azurin in the solid-state Au/protein/Au junction by monitoring protein vibrations during current transport under applied bias, up to ≈1 GV m
⁻¹, by electrical detection of inelastic electron transport effects. Characteristic vibrational modes, such as C-H stretching, amide (N-H) bending, and A-S (of the bonds that connect the protein to an Au electrode), are not found to change noticeably up to 1.0 V. At >1.0 V, the N-H bending and C-H stretching inelastic features have disappeared, while the Au-S features persist till ≈2 V, i.e., the proteins remain Au bound. Three possible causes for the disappearance of the N-H and C-H inelastic features at high bias, namely, i) resonance transport, ii) metallic filament formation, and iii) bond rupture leading to structural changes in the protein are proposed and tested. The results support the last option and indicate that spectrally resolved inelastic features can serve to monitor in operando structural stability of biological macromolecules while they serve as electronic current conduit.

We report on charge transport across single short peptides using the Mechanically Controlled Break Junction (MCBJ) method. We record thousands of electron transport events across single-molecule junctions and with an unsupervised machine learning algorithm, we identify several classes of traces with multifarious conductance values that may correspond to different peptide conformations. Data analysis shows that very short peptides, which are more rigid, show conductance plateaus at low conductance values of about 10-3G0 and below, with G0 being the conductance quantum, whereas slightly longer, more flexible peptides also show plateaus at higher values. Fully stretched peptide chains exhibit conductance values that are of the same order as that of alkane chains of similar length. The measurements show that in the case of short peptides, different compositions and molecular lengths offer a wide range of junction conformations. Such information is crucial to understand mechanism(s) of charge transport in and across peptide-based biomolecules. 

We observe reversible, bias-induced switching of conductance via a blue copper protein azurin mutant, N42C Az, with a nearly 10-fold increase at |V| > 0.8 V than at lower bias. No such switching is found for wild-type azurin, WT Az, up to |1.2 V|, beyond which irreversible changes occur. The N42C Az mutant will, when positioned between electrodes in a solid-state AuproteinAu junction, have an orientation opposite that of WT Az with respect to the electrodes. Current(s) via both proteins are temperature-independent, consistent with quantum mechanical tunneling as dominant transport mechanism. No noticeable difference is resolved between the two proteins in conductance and inelastic electron tunneling spectra at

Retinal proteins play significant roles in light-induced protons/ions transport across the cell membrane. A recent studied retinal protein, gloeobacter rhodopsin (gR), functions as a proton pump, and binds the carotenoid salinixanthin (sal) in addition to the retinal chromophore. We have studied the interactions between the two chromophores as reflected in the circular dichroism (CD) spectrum of gR complex. gR exhibits a weak CD spectrum but following binding of sal, it exhibits a significant enhancement of the CD bands. To examine the CD origin, we have substituted the retinal chromophore of gR by synthetic retinal analogues, and have concluded that the CD bands originated from excitonic interaction between sal and the retinal chromophore as well as the sal chirality induced by binding to the protein. Temperature increase significantly affected the CD spectra, due to vanishing of excitonic coupling. A similar phenomenon of excitonic interaction lose between chromophores was recently reported for a photosynthetic pigment-protein complex (Nature Commmun, 9, 2018, 99). We propose that the excitonic interaction in gR is weaker due to protein conformational alterations. The excitonic interaction is further diminished following reduction of the retinal protonated Schiff base double bond. Furthermore, the intact structure of the retinal ring is necessary for obtaining the excitonic interaction.

Successful integration of proteins in solid-state electronics requires contacting them in a non-invasive fashion, with a solid conducting surface for immobilization as one such contact. The contacts can affect and even dominate the measured electronic transport. Often substrates, substrate treatments, protein immobilization, and device geometries differ between laboratories. Thus the question arises how far results from different laboratories and platforms are comparable and how to distinguish genuine protein electronic transport properties from platform-induced ones. We report a systematic comparison of electronic transport measurements between different laboratories, using all commonly used large-area schemes to contact a set of three proteins of largely different types. Altogether we study eight different combinations of molecular junction configurations, designed so that A(geo) of junctions varies from 10(5) to 10(-3) mu m(2). Although for the same protein, measured with similar device geometry, results compare reasonably well, there are significant differences in current densities (an intensive variable) between different device geometries. Likely, these originate in the critical contact-protein coupling (similar to contact resistance), in addition to the actual number of proteins involved, because the effective junction contact area depends on the nanometric roughness of the electrodes and at times, even the proteins may increase this roughness. On the positive side, our results show that understanding what controls the coupling can make the coupling a design knob. In terms of extensive variables, such as temperature, our comparison unanimously shows the transport to be independent of temperature for all studied configurations and proteins. Our study places coupling and lack of temperature activation as key aspects to be considered in both modeling and practice of protein electronic transport experiments.

Solid-state electronic transport (ETp) via the electron-transfer copper protein azurin (Az) was measured in Au/Az/Au junction configurations down to 4 K, the lowest temperature for solid-state protein-based junctions. Not only does lowering the temperature help when observing fine features of electronic transport, but it also limits possible electron transport mechanisms. Practically, wire-bonded devices-on-chip, carrying Az-based microscopic junctions, were measured in liquid He, minimizing temperature gradients across the samples. Much smaller junctions, in conducting-probe atomic force microscopy measurements, served, between room temperature and the protein's denaturation temperature (similar to 323 K), to check that conductance behavior is independent of device configuration or contact nature and thus is a property of the protein itself. Temperature-independent currents were observed from similar to 320 to 4 K. The experimental results were fitted to a single-level Landauer model to extract effective energy barrier and electrode-molecule coupling strength values and to compare data sets. Our results strongly support that quantum tunneling, rather than hopping, dominates ETp via Az.

A sample-type protein monolayer, that can be a stepping stone to practical devices, can behave as an electrically driven switch. This feat is achieved using a redox protein, cytochrome C (CytC), with its heme shielded from direct contact with the solid-state electrodes. Ab initio DFT calculations, carried out on the CytC-Au structure, show that the coupling of the heme, the origin of the protein frontier orbitals, to the electrodes is sufficiently weak to prevent Fermi level pinning. Thus, external bias can bring these orbitals in and out of resonance with the electrode. Using a cytochrome C mutant for direct S-Au bonding, approximately 80 % of the Au-CytC-Au junctions show at greater than 0.5 V bias a clear conductance peak, consistent with resonant tunneling. The on-off change persists up to room temperature, demonstrating reversible, bias-controlled switching of a protein ensemble, which, with its built-in redundancy, provides a realistic path to protein-based bioelectronics.

We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose-synthesized, peptide amphiphiles, C-10-(Asp)(5) and C-10-(Lys)(5). Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C-10-(Asp)(5) are conjugated (a) via a water-soluble, penta-Lys mediator or (b) to micelles containing the C-10-(Lys)(5) peptide amphiphile. Both routes lead to phase separation in the form of oil-rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light-driven proton pump King Sejong 1-2 (KS1-2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.

Retinal proteins' biological activity is triggered by the retinal chromophore's light absorption, which initiates a photocycle. However, the mechanism by which retinal light excitation induces the protein's response is not completely understood. Recently, two new retinal proteins were discovered, namely, King Sejong 1-2 (KS1-2) and Nonlabens (Donghaeana) dokdonensis (DDR2), which exhibit H+ and Na+ pumping activities, respectively. To pinpoint whether protein conformation alterations can be achieved without light-induced retinal C-13=C-14 double-bond isomerization, we utilized the hydroxylamine reaction, which cleaves the protonated Schiff base bond through which the retinal chromophore is covalently bound to the protein. The reaction is accelerated by light even though the cleavage is not a photochemical reaction. Therefore, the cleavage reaction may serve as a tool to detect protein conformation alterations. We discovered that in both KS1-2 and DDR2, the hydroxylamine reaction is light accelerated, even in artificial pigments derived from synthetic retinal in which the crucial C-13=C-14 double-bond isomerization is prevented. Therefore, we propose that in both proteins the light-induced retinal charge redistribution taking place in the retinal excited state polarizes the protein, which, in turn, triggers protein conformation alterations. A further general possible application of the present finding is associated with other photoreceptor proteins having retinal or other non-retinal chromophores whose light excitation may affect the protein conformation.

In microbial rhodopsins (also called retinal proteins), the retinal chromophore is used for harvesting light. A carotenoid molecule has been reported to complement the retinal as light harvesting antenna in bacterial retinal proteins, although examples are scarce. In this paper, we present the formation of a novel antenna complex between thermophilic rhodopsin (TR) and the carotenoid salinixanthin (Sal). The complex formation and its structure were studied using UV-visible absorption as well as circular dichroism (CD) spectroscopies. Our studies indicate that the complex is formed in both the trimeric and monomeric forms of TR. CD spectroscopy suggests that excitonic coupling takes place between retinal and Sal. The binding of Sal with artificial TR pigments derived from synthetic retinal analogues further supports the contribution of the retinal chromophore to the CD spectrum. These studies further support the possibility of interaction between the 4-keto ring of the Sal and the retinal in TR-Sal complexes. Temperature-dependent CD spectra indicate that the positive band (ca. 482 nm) of the bisignate CD spectra of the studied complexes originates from the contribution of excitonic coupling and induced chirality of Sal in the protein binding site. The presence of a relatively smaller glycine residue in the vicinity of the retinal chromophore in TR is proposed to be crucial for binding with Sal. The results are expected to shed light on the mechanism of retinal carotenoid interactions in other biological systems.

Different batches of Si wafers with nominally the same specifications were found to respond differently to identical chemical surface treatments aimed at regrowing Si oxide on them. We found that the oxides produced on different batches of wafer differ electrically, thereby affecting solid-state electron transport (ETp) via protein films assembled on them. These results led to the another set of experiments, where we studied this phenomenon using two distinct chemical methods to regrow oxides on the same batch of Si wafers. We have characterized the surfaces of the regrown oxides and of monolayers of linker molecules that connect proteins with the oxides and examined ETp via ultrathin layers of the protein bacteriorhodopsin, assembled on them. Our results illustrate the crucial role of (near) surface charges on the substrate in defining the ETp characteristics across the proteins. This is expressed most strikingly in the observed current's temperature dependences, and we propose that these are governed by the electrostatic landscape at the electrode-protein interface rather than by intrinsic protein properties. This study's major finding, relevant to protein bioelectronics, is that protein-electrode coupling in junctions is a decisive factor in ETp across them. Hence,surface electrostatics can create a barrier that dominates charge transport and controls the transport mode across the junction. Our findings' wider importance lies in their relevance to hybrid junctions of Si with (polyelectrolyte) biomolecules, a likely direction for future bioelectronics. A remarkable corollary of presented results is that once an electron is injected into the protein, transport within the proteins is so efficient that it does not encounter a measurable barrier down to 160 K.

The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are.

Making biomolecular electronics a reality will require control over charge transport across biomolecules. Here we show that chemical modulation of the coupling between one of the electronic contacts and the biomolecules in a solid-state junction allows controlling electron transport (ETp) across the junction. Employing the protein azurin (Az), we achieve such modulation as follows: Az is covalently bound by Au-S bonding to a lithographically prepared Au electrode (Au-Az). Au nanowires (AuNW) onto which linker molecules, with free carboxylic group, are bound via Au-S bonds serve as top electrode. Current voltage plots of AuNW-linkerCOOH//Az-Au junctions have been shown earlier to exhibit step-like features, due to resonant tunneling through discrete Az energy levels. Forming an amide bond between the free carboxylic group of the AuNW-bound linker and Az yields AuNW-linkerCO-NH-Az-Au junctions. This Az-linker bond switches the ETp mechanism from resonant to off-resonant tunneling. By varying the extent of this amide bonding, the current voltage dependence can be controlled between these two mechanisms, thus providing a platform for altering and controlling the ETp mechanism purely by chemical modification in a two-terminal device, i.e., without a gate electrode. Using results from conductance, including the energy barrier and electrode molecule coupling parameters extracted from current voltage fitting and normalized differential conductance analysis and from inelastic-electron-tunneling and photoelectron spectroscopies, we determine the Az frontier orbital energies, with respect to the Au Fermi level, for four junction configurations, differing only in electrode-protein coupling. Our approach and findings open the way to both qualitative and quantitative control of biomolecular electronic junctions.

Metalloproteins, proteins containing a transition metal ion cofactor, are electron transfer agents that perform key functions in cells. Inspired by this fact, electron transport across these proteins has been widely studied in solid-state settings, triggering the interest in examining potential use of proteins as building blocks in bioelectronic devices. Here, we report results of low-temperature (10 K) electron transport measurements via monolayer junctions based on the blue copper protein azurin (Az), which strongly suggest quantum tunneling of electrons as the dominant charge transport mechanism. Specifically, we show that, weakening the protein-electrode coupling by introducing a spacer, one can switch the electron transport from off-resonant to resonant tunneling. This is a consequence of reducing the electrode's perturbation of the Cu(II)localized electronic state, a pattern that has not been observed before in protein-based junctions. Moreover, we identify vibronic features of the Cu(II) coordination sphere in transport characteristics that show directly the active role of the metal ion in resonance tunneling. Our results illustrate how quantum mechanical effects may dominate electron transport via protein-based junctions.

We review the status of protein-based molecular electronics. First, we define and discuss fundamental concepts of electron transfer and transport in and across proteins and proposed mechanisms for these processes. We then describe the immobilization of proteins to solid-state surfaces in both nanoscale and macroscopic approaches, and highlight how different methodologies can alter protein electronic properties. Because immobilizing proteins while retaining biological activity is crucial to the successful development of bioelectronic devices, we discuss this process at length. We briefly discuss computational predictions and their connection to experimental results. We then summarize how the biological activity of immobilized proteins is beneficial for bioelectronic devices, and how conductance measurements can shed light on protein properties. Finally, we consider how the research to date could influence the development of future bioelectronic devices.

Over the past few decades, the structure, functions, properties, and molecular mechanisms of retinal proteins have been studied extensively. The newly studied retinal protein Gloeobacter rhodopsin (gR) acts as a light-driven proton pump, transferring a proton from the cytoplasmic region to the extracellular region of a cell following light absorption. It was previously shown that gR can bind the carotenoid salinixanthin (sal). In the present study, we report the effect of pH on the binding of retinal to the apo-protein of gR, in the presence and absence of sal, to form the gR pigment. We found that binding at different pH levels reflects the titration of two different protein residues, one at the lower pK(a) 3.5 and another at the higher pK(a) 8.4, that affect the pigment's formation. The maximum amount of pigment was formed at pH 5, both with and without the presence of sal. The introduction of sal accelerates the rate of pigment formation by a factor of 190. Furthermore, it is suggested that occupation of the binding site by the retinal chromophore induces protein conformational alterations which in turn affect the carotenoid conformation, which precedes the formation of the retinal-protein covalent bond. Our examination of synthetic retinal analogues in which the ring structure was modified revealed that, in the absence of sal, the retinal ring structure affects the rate of pigment formation and that the intact structure is needed for efficient pigment formation. However, the presence of sal abolishes this effect, and all-trans retinal and its modified ring analogues bind at a similar rate.

Ultrafast photochemistry of pharaonis halorhodopsin (p-HR) in the intact membrane of Natronomonas pharaonis has been studied by photoselective femtosecond pump-hyperspectral probe spectroscopy with high time resolution. Two variants of this sample were studied, one with wild-type retinal prosthetic groups and another after shifting the retinal absorption deep into the blue range by reducing the Schiff base linkage, and the results were compared to a previous study on detergent-solubilized p-HR. This comparison shows that retinal photoisomerization dynamics is identical in the membrane and in the solubilized sample. Selective photoexcitation of bacterioruberin, which is associated with the protein in the native membrane, in wild-type and reduced samples, demonstrates conclusively that unlike the carotenoids associated with some bacterial retinal proteins the carrotenoid in p-HR does not act as a light-harvesting antenna. (Graph Presented).

Lake Baikal is the deepest and one of the most ancient lakes in the world. Its unique ecology has resulted in the colonization of a diversity of depth habitats by a unique fauna that includes a group of teleost fish of the sub-order Cottoidei. This relatively recent radiation of cottoid fishes shows a gradual blue-shift in the wavelength of the absorption maximum of their visual pigments with increasing habitat depth. Here we combine homology modeling and quantum chemical calculations with experimental in vitro measurements of rhodopsins to investigate dim-light adaptation. The calculations, which were able to reproduce the trend of observed absorption maxima in both A1 and A2 rhodopsins, reveal a Barlow-type relationship between the absorption maxima and the thermal isomerization rate suggesting a link between the observed blue-shift and a thermal noise decrease. A Nakanishi point-charge analysis of the electrostatic effects of non-conserved and conserved amino acid residues surrounding the rhodopsin chromophore identified both close and distant sites affecting simultaneously spectral tuning and visual sensitivity. We propose that natural variation at these sites modulate both the thermal noise and spectral shifting in Baikal cottoid visual pigments resulting in adaptations that enable vision in deep water light environments.

The group of homoiochlorophyllous resurrection plants evolved the unique capability to survive severe drought stress without dismantling the photosynthetic machinery. This implies that they developed efficient strategies to protect the leaves from reactive oxygen species (ROS) generated by photosynthetic side reactions. These strategies, however, are poorly understood. Here, we performed a detailed study of the photosynthetic machinery in the homoiochlorophyllous resurrection plant Craterostigma pumilum during dehydration and upon recovery from desiccation. During dehydration and rehydration, C.pumilum deactivates and activates partial components of the photosynthetic machinery in a specific order, allowing for coordinated shutdown and subsequent reinstatement of photosynthesis. Early responses to dehydration are the closure of stomata and activation of electron transfer to oxygen accompanied by inactivation of the cytochrome b(6)f complex leading to attenuation of the photosynthetic linear electron flux (LEF). The decline in LEF is paralleled by a gradual increase in cyclic electron transport to maintain ATP production. At low water contents, inactivation and supramolecular reorganization of photosystem II becomes apparent, accompanied by functional detachment of light-harvesting complexes and interrupted access to plastoquinone. This well-ordered sequence of alterations in the photosynthetic thylakoid membranes helps prepare the plant for the desiccated state and minimize ROS production. Significance Statement Dehydration in plants is accompanied by an increase in reactive oxygen species. The resurrection plant Craterostigma pumilum avoids damage by reactive oxygen species during dehydration. A mechanism is shown in this study that is based on a well-ordered combination of common changes in the photosynthetic machinery.

Charge migration for electron transfer via the polypeptide matrix of proteins is a key process in biological energy conversion and signaling systems. It is sensitive to the sequence of amino acids composing the protein and, therefore, offers a tool for chemical control of charge transport across biomaterial-based devices. We designed a series of linear oligoalanine peptides with a single tryptophan substitution that acts as a "dopant," introducing an energy level closer to the electrodes' Fermi level than that of the alanine homopeptide. We investigated the solid-state electron transport (ETp) across a selfassembled monolayer of these peptides between gold contacts. The single tryptophan "doping" markedly increased the conductance of the peptide chain, especially when its location in the sequence is close to the electrodes. Combining inelastic tunneling spectroscopy, UV photoelectron spectroscopy, electronic structure calculations by advanced density-functional theory, and dc current-voltage analysis, the role of tryptophan in ETp is rationalized by charge tunneling across a heterogeneous energy barrier, via electronic states of alanine and tryptophan, and by relatively efficient direct coupling of tryptophan to a Au electrode. These results reveal a controlled way of modulating the electrical properties of molecular junctions by tailormade "building block" peptides.

Previous studies have shown that the gas-phase fragmentation of the retinal chromophore after S0-S1 photoexcitation results in a prominent fragment of mass 248 which cannot be explained by the cleavage of any single bond along the polyene chain. It was therefore theorized that the fragmentation mechanism involves a series of isomerizations and cyclization processes, and two mechanisms for these processes were suggested. Here we used isotope labeling MS-MS to provide conclusive support for the fragmentation mechanism suggested by Coughlan et al. (J. Phys. Chem. Lett. 2014, 5, 3195).

A vertical nanogap device (VND) structure comprising all-silicon contacts as electrodes for the investigation of electronic transport processes in bioelectronic systems is reported. Devices were fabricated from silicon-on-insulator substrates whose buried oxide (SiO₂) layer of a few nanometers in thickness is embedded within two highly doped single crystalline silicon layers. Individual VNDs were fabricated by standard photolithography and a combination of anisotropic and selective wet etching techniques, resulting in p⁺ silicon contacts, vertically separated by 4 or 8 nm, depending on the chosen buried oxide thickness. The buried oxide was selectively recess-etched with buffered hydrofluoric acid, exposing a nanogap. For verification of the devices' electrical functionality, gold nanoparticles were successfully trapped onto the nanogap electrodes' edges using AC dielectrophoresis. Subsequently, the suitability of the VND structures for transport measurements on proteins was investigated by functionalizing the devices with cytochrome c protein from solution, thereby providing non-destructive, permanent semiconducting contacts to the proteins. Current-voltage measurements performed after protein deposition exhibited an increase in the junctions' conductance of up to several orders of magnitude relative to that measured prior to cytochrome c immobilization. This increase in conductance was lost upon heating the functionalized device to above the protein's denaturation temperature (80 °C). Thus, the VND junctions allow conductance measurements which reflect the averaged electronic transport through a large number of protein molecules, contacted in parallel with permanent contacts and, for the first time, in a symmetrical Si-protein-Si configuration.

The present work studies the mechanism of light induced protein conformational changes in the over-expressed mutant of halorhodopsin (phR) from Natronomonas pharaonis. The catalytic effect of light is reflected in accelerating hydroxyl amine reaction rate of light adapted phR. Light catalysis was detected in native phR but also in artificial pigments derived from tailored retinal analogs locked at the crucial C₁₃=C₁₄ double bond. It is proposed that the photoexcited retinal chromophore induces protein concerted motion that decreases the energy gap between reactants ground and transition states. This energy gap is overcome by coupling to specific protein vibrations. Surprisingly, the rate constants show unusual decreasing trend following temperature increase both for native and artificial pigments.

Surprisingly efficient solid-state electron transport has recently been demonstrated through "dry" proteins (with only structural, tightly bound H₂O left), suggesting proteins as promising candidates for molecular (bio)electronics. Using inelastic electron tunneling spectroscopy (IETS), we explored electron-phonon interaction in metal/protein/metal junctions, to help understand solid-state electronic transport across the redox protein azurin. To that end an oriented azurin monolayer on Au is contacted by soft Au electrodes. Characteristic vibrational modes of amide and amino acid side groups as well as of the azurin-electrode contact were observed, revealing the azurin native conformation in the junction and the critical role of side groups in the charge transport. The lack of abrupt changes in the conductance and the line shape of IETS point to far off-resonance tunneling as the dominant transport mechanism across azurin, in line with previously reported (and herein confirmed) azurin junctions. The inelastic current and hence electron-phonon interaction appear to be rather weak and comparable in magnitude with the inelastic fraction of tunneling current via alkyl chains, which may reflect the known structural rigidity of azurin.

Many novel applications in bioelectronics rely on the interaction between biomolecules and electronically conducting substrates. However, crucial knowledge about the relation between electronic transport via peptides and their amino-acid composition is still absent. Here, we report results of electronic transport measurements via several homopeptides as a function of their structural properties and temperature. We demonstrate that the conduction through the peptide depends on its length and secondary structure as well as on the nature of the constituent amino acid and charge of its residue. We support our experimental observations with high-level electronic structure calculations and suggest off-resonance tunneling as the dominant conduction mechanism via extended peptides. Our findings indicate that both peptide composition and structure can affect the efficiency of electronic transport across peptides.

Searching for novel hybrid electrocatalysts with high activity and strong durability for a direct electrochemical hydrogen evolution reaction (HER) is extremely desirable but still remains a significant challenge. Herein, we report a novel solid carbon cloth-supported hybrid nano-bio electrocatalyst, decorated with Ag nanoparticles and proton-pumping bacteriorhodopsin (bR) (Ag/bR/CP) that were prepared by in situ electroless deposition and vesicle fusion technology, respectively. When applied as a hydrogen evolution cathode, the Ag/bR/CP shows a low onset overpotential of 63 mV, good durability (no detectable change in its catalytic activity for up to 1000 cycles in alkaline media), and enhanced HER performance under 550 nm irradiation, attributed to the activation of Ag and synergistic effects following light absorption, demonstrated by photoelectrochemical measurements.

The role of the electron spin in chemistry and biology has received much attention recently owing to to the possible electromagnetic field effects on living organisms and the prospect of using molecules in the emerging field of spintronics. Recently the chiral-induced spin selectivity effect was observed by electron transmission through organic molecules. In the present study, we demonstrated the ability to control the spin filtering of electrons by light transmitted through purple membranes containing bacteriorhodopsin (bR) and its D96N mutant. The spin-dependent electrochemical cyclic voltammetry (CV) and chronoamperometric measurements were performed with the membranes deposited on nickel substrates. High spin-dependent electron transmission through the membranes was observed; however, after the samples were illuminated by 532 nm light, the spin filtering in the D96N mutant was dramatically reduced whereas the light did not have any effect on the wild-type bR. Beyond demonstrating spin-dependent electron transmission, this work also provides an interesting insight into the relationship between the structure of proteins and spin filtering by conducting electrons.

We describe two alternative and complementary purification methods for halorhodopsin and bacteriorhodopsin. The first relies on a unique form of detergent micelles which we have called engineered-micelles. These are specifically conjugated in the presence of [hydrophobic chelator:Fe(2+)] complexes and form detergent aggregates into which membrane proteins partition, but hydrophilic water-soluble proteins do not. The approach was tested on the membrane protein, bacteriorhodopsin (bR), with five non-ionic detergents (OG, OTG, NG, DM, and DDM), commonly used in purification and crystallization of membrane proteins, in combination with the commercially available bathophenanthroline or with one of the three synthesized phenanthroline derivatives (Phen-C10, Phen-C8 and Phen-C6). Our results show that bR is extracted efficiently (60-86%) and directly from its native membrane into diverse detergent aggregates with preservation of its native conformation, while 90-95% of an artificial contaminating background is excluded. For implementation of the second method, based on engineered-membranes, the use of detergents, which in some cases may produce protein denaturation, is not required at all. Protein-containing membranes are conjugated via the same hydrophobic [chelator:metal ion] complexes but maintain the membrane protein in its native bilayer environment throughout the process. This method is demonstrated on the membrane protein halorhodopsin from Natronomonas pharaonis (phR) and leads to good recovery yields (74-89%) and removal of >85% of artificial background impurities while preserving the native state of phR. The detailed structure of the hydrophobic chelator used has been found to have a marked effect on the purity and yield of both methods.

Optogenetic tools have become indispensable in neuroscience to stimulate or inhibit excitable cells by light. Channelrhodopsin-2 (ChR2) variants have been established by mutating the opsin backbone or by mining related algal genomes. As an alternative strategy, we surveyed synthetic retinal analogues combined with microbial rhodopsins for functional and spectral properties, capitalizing on assays in C. elegans, HEK cells and larval Drosophila. Compared with all-trans retinal (ATR), Dimethylamino-retinal (DMAR) shifts the action spectra maxima of ChR2 variants H134R and H134R/T159C from 480 to 520 nm. Moreover, DMAR decelerates the photocycle of ChR2(H134R) and (H134R/T159C), thereby reducing the light intensity required for persistent channel activation. In hyperpolarizing archaerhodopsin-3 and Mac, naphthyl-retinal and thiophene-retinal support activity alike ATR, yet at altered peak wavelengths. Our experiments enable applications of retinal analogues in colour tuning and altering photocycle characteristics of optogenetic tools, thereby increasing the operational light sensitivity of existing cell lines or transgenic animals.Errata: In this Article, there are errors in the formulae depicted in Fig. 1a that were introduced during the production process.

Potential future use of bacteriorhodopsin (bR) as a solid-state electron transport (ETp) material requires the highest possible active protein concentration. To that end we prepared stable monolayers of protein-enriched bR on a conducting HOPG substrate by lipid depletion of the native bR. The ETp properties of this construct were then investigated using conducting probe atomic force microscopy at low bias, both in the ground dark state and in the M-like intermediate configuration, formed upon excitation by green light. Photoconductance modulation was observed upon green and blue light excitation, demonstrating the potential of these monolayers as optoelectronic building blocks. To correlate protein structural changes with the observed behavior, measurements were made as a function of pressure under the AFM tip, as well as humidity. The junction conductance is reversible under pressure changes up to ∼300 MPa, but above this pressure the conductance drops irreversibly. ETp efficiency is enhanced significantly at >60% relative humidity, without changing the relative photoactivity significantly. These observations are ascribed to changes in protein conformation and flexibility and suggest that improved electron transport pathways can be generated through formation of a hydrogen-bonding network.

Electronic coupling to electrodes, G, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron- mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and -130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases G, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, Ea, of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower Ea values (from high-temperature data) and higher conductance at low temperatures (in the temperatureindependent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width).

Spin-dependent photoelectron transmission and spin-dependent electrochemical studies were conducted on purple membrane containing bacteriorhodopsin (bR) deposited on gold, aluminum/aluminum-oxide, and nickel substrates. The result indicates spin selectivity in electron transmission through the membrane. Although the chiral bR occupies only about 10% of the volume of the membrane, the spin polarization found is on the order of 15%. The electrochemical studies indicate a strong dependence of the conduction on the protein's structure. Denaturation of the protein causes a sharp drop in the conduction through the membrane.

A novel method for purifying membrane proteins is presented. The approach makes use of engineered micelles composed of a nonionic detergent, β-octylglucoside, and a hydrophobic metal chelator, bathophenanthroline. Via the chelators, the micelles are specifically conjugated, i.e., tethered, in the presence of Fe²⁺ ions, thereby forming micellar aggregates which provide the environment for separation of lipid-soluble membrane proteins from water-soluble proteins. The micellar aggregates (here imaged by cryo-transmission electron microscopy) successfully purify the light driven proton pump, bacteriorhodopsin (bR), from E. coli lysate. Purification takes place within 15 min and can be performed both at room temperature and at 4 C. More than 94% of the water-soluble macromolecules in the lysate are excluded, with recovery yields of the membrane protein ranging between 74% and 85%. Since this approach does not require precipitants, high concentrations of detergent to induce micellar aggregates, high temperature, or changes in pH, it is suggested that it may be applied to the purification of a wide variety of membrane proteins.

Photochemistry of bacteriorhodopsin (bR), anabaena sensory rhodopsin (ASR), and all-trans retinal protonated Schiff base (RPSB) in ethanol is followed with femtosecond pump-hyperspectral near-IR (NIR) probe spectroscopy. This is the first systematic probing of retinal protein photochemistry in this spectral range. Stimulated emission of the proteins is demonstrated to extend deep into the NIR, and to decay on the same characteristic time scales previously determined by visible probing. No signs of a transient NIR absorption band above λ_pr > 1.3 μm, which was recently reported and is verified here for the RPSB in solution, is observed in either protein. This discrepancy demonstrates that the protein surroundings change photochemical traits of the chromophore significantly, inducing changes either in the energies or couplings of photochemically relevant electronic excited states. In addition, low-frequency and heavily damped spectral modulations are observed in the NIR signals of all three systems up to 1.4 μm. By background subtraction and Fourier analysis they are shown to resemble wave packet signatures in the visible, stemming from multiple vibrational modes and by analogy are assigned to torsional wave packets in the excited state of the retinal chromophore. Differences in the vibrational frequencies between the three samples and the said discrepancy in transient spectra are discussed in terms of opsin effects on the RPSB electronic structure.

The ultrafast spectroscopic investigation of novel retinal proteins challenges existing notions concerning the course of primary events in these natural photoreceptors. We review two illustrations here. The first demonstrates that changes in the initial retinal configuration can alter the duration of photochemistry by nearly an order of magnitude in Anabaena sensory rhodopsin, making it as rapid as the ballistic photoisomerization in visual pigments. This prompted a reinvestigation of the much studied bacteriorhodopsin, leading to a similar trend as well, contrary to earlier reports. The second involves the study of xanthorhodopsin, an archaeal proton pump that includes an attached light-harvesting carotenoid. Pump-probe experiments demonstrate the efficient transfer of energy from carotenoid to retinal, providing a first glimpse at a cooperative multichromophore function, which is probably characteristic of many other proteins as well. Finally, we discuss measures required to advance our knowledge from kinetics to mode-specific dynamics concerning this expanding family of biological photoreceptors.

A strategy for clustering of native lipid membranes is presented. It relies on the formation of complexes between hydrophobic chelators embedded within the lipid bilayer and metal cations in the aqueous phase, capable of binding two (or more) chelators simultaneously Fig. 1. We used this approach with purple membranes containing the light driven proton pump protein bacteriorhodopsin (bR) and showed that patches of purple membranes cluster into mm sized aggregates and that these are stable for months when incubated at 19°C in the dark. The strategy may be general since four different hydrophobic chelators (1,10-phenanthroline, bathophenanthroline, Phen-C10, and 8-hydroxyquinoline) and various divalent cations (Ni²⁺, Zn²⁺, Cd²⁺, Mn²⁺, and Cu²⁺) induced formation of membrane clusters. Moreover, the absolute requirement for a hydrophobic chelator and the appropriate metal cations was demonstrated with light and atomic force microscopy (AFM); the presence of the metal does not appear to affect the functional state of the protein. The potential utility of the approach as an alternative to assembled lipid bilayers is suggested.

Electrons can migrate via proteins over distances that are considered long for nonconjugated systems. The nanoscale dimensions of proteins and their enormous structural and chemical flexibility makes them fascinating subjects for exploring their electron transport (ETp) capacity. One particularly attractive direction is that of tuning their ETp efficiency by "doping" them with small molecules. Here we report that binding of retinoate (RA) to human serum albumin (HSA) increases the solid-state electronic conductance of a monolayer of the protein by >2 orders of magnitude for RA/HSA ≤ 3. Temperature-dependent ETp measurements show the following with increasing RA/HSA: (a) The temperature-independent current magnitude of the low-temperature (300-fold), suggesting a decrease in the distance-decay constant of the process. (b) The activation energy of the thermally activated regime (>190 K) decreases from 220 meV (RA/HSA = 0) to 70 meV (RA/HSA ≤ 3).

Absorption of light by the visual pigment rhodopsin triggers a rapid cis-trans photoisomerization of its retinal chromophore and a series of conformational changes in both the retinal and protein. The largest structural change is an outward tilt of transmembrane helix H6 that increases the separation of the intracellular ends of H6 and H3 and opens up the G-protein binding site. In the dark state of rhodopsin, Glu247 at the intracellular end of H6 forms a salt bridge with Arg135 on H3 to tether H6 in an inactive conformation. The Arg135-Glu247 interaction is broken in the active state of the receptor, and Arg135 is then stabilized by interactions with Tyr223, Met257, and Tyr306 on helices H5, H6, and H7, respectively. To address the mechanism of H6 motion, solid-state NMR measurements are undertaken of Metarhodopsin I (Meta I), the intermediate preceding the active Metarhodopsin II (Meta II) state of the receptor. ¹³C NMR dipolar recoupling measurements reveal an interhelical contact of ¹³Cχ-Arg135 with ¹³Cμ- Met257 in Meta I but not with ¹³Cχ-Tyr223 or ¹³Cχ- Tyr306. These observations suggest that helix H6 has rotated in the formation of Meta I but that structural changes involving helices H5 and H7 have not yet occurred. Together, our results provide insights into the sequence of events leading up to the outward motion of H6, a hallmark of G protein-coupled receptor activation.

Photochemistry in retinal proteins (RPs) is determined both by the properties of the retinal chromophore and by its interactions with the surrounding protein. The initial retinal configuration, and the isomerization coordinates active in any specific protein, must be important factors influencing the course of photochemistry. This is illustrated by the vast differences between the photoisomerization dynamics in visual pigments which start 11-cis and end all-trans, and those observed in microbial ion pumps and sensory rhodopsins which start all-trans and end in a 13-cis configuration. However, isolating these factors is difficult since most RPs accommodate only one active stable ground-state configuration. Anabaena sensory rhodopsin, allegedly functioning in cyanobacteria as a wavelength sensor, exists in two stable photoswitchable forms, containing all-trans and 13-cis retinal isomers, at a wavelength-dependent ratio. Using femtosecond spectroscopy, and aided by extraction of coherent vibrational signatures, we show that cis-to-trans photoisomerization, as in visual pigments, is ballistic and over in a fraction of a picosecond, while the reverse is nearly 10 times slower and kinetically reminiscent of other microbial rhodopsins. This provides a new test case for appreciating medium effects on primary events in RPs.

Excited state dynamics of native Xanthorhodopsin (XR), of an XR sample with a reduced prosthetic group, and of the associated Carotenoid (CAR) salinixanthin (SX) in ethanol were investigated by hyperspectral Near Infrared (NIR) probing. Global kinetic analysis shows that: (1) unlike the transient spectra recorded in the visible, fitting of the NIR data requires only two phases of exponential spectral evolution, assigned to internal conversion from S₂ → S₁ and from S₁ → S₀ of the carotene. (2) The rate of the internal conversion from S₂ → S₁ in the reduced sample is well fit with a decay time of 130 fs, significantly longer than in XR and in SX, both of which are well fit with τ ≈ 100 fs. This increased lifetime is consistent with a ∼30% efficiency of ET from SX to retinal in XR. (3) S₁ of salinixanthin is verified to lie ∼12700 cm^-1 above the ground electronic surface, excluding its involvement in the retinal sensitization in XR. (4) The oscillator strength of the S₁ → S₂ transition is determined to be no more than 0.16, despite its symmetry allowedness. (5) No long lived NIR absorbance decay assignable to the carotenoid S* state was detected in any of the samples. Inconsistencies concerning previously determined S₂ lifetimes and kinetic schemes used to model these data are discussed.

Second-harmonic generation (SHG) by membrane-incorporated probes is a nonlinear optical signal that is voltage-sensitive and the basis of a sensitive method for imaging membrane potential. The voltage dependence of SHG by four different probes, three retinoids (all-trans retinal), and two new retinal analogs, 3-methyl-7-(4́-dimethylamino-phenyl)- 2,4,6-heptatrienal (AR-3) and 3,7-dimethyl-9-(4́-dimethylamino-phenyl)-2,4,6,8-nonatetraenal (AR-4), and a styryl dye (FM4-64), were compared in HEK-293 cells. Results were analyzed by fitting data with an expression based on an electrooptic mechanism for SHG, which depends on the complex-valued first- and second-order nonlinear electric susceptibilities (x2 and x3) of the probe. This gave values for the voltage sensitivity at the cell's resting potential, the voltage where the SHG is minimal, and the amplitude of the signal at that voltage for each of the four compounds. These measures show that x2 and x3 are complex numbers for all compounds except all-trans retinal, consistent with the proximities of excitation and/or emission wavelengths to molecular resonances. Estimates of probe orientation and location in the membrane electric field show that, for the far-fromresonance case, the shot noise-limited signal/noise ratio depends on the location of the probe in the membrane, and on x3 but not on x2.

Protein structures can facilitate long-range electron transfer in solution. But a fundamental question remains: can these structures also serve as solid-state electronic conductors? Answering this question requires methods for studying conductivity of the "dry" protein (which only contains tightly bound structured water molecules) sandwiched between two electronic conductors in a solid-state type configuration. If successful, such systems could serve as the basis for future, bioinspired electronic device technology. In this Account, we survey, analyze, and compare macroscopic and nanoscopic (scanning probe) solid-state conductivities of proteins, noting the inherent constraints of each of these, and provide the first status report on this research area. This analysis shows convincing evidence that "dry" proteins pass orders of magnitude higher currents than saturated molecules with comparable thickness and that proteins with known electrical activity show electronic conductivity, nearly comparable to that of conjugated molecules ("wires"). These findings suggest that the structural features of proteins must have elements that facilitate electronic conductivity, even if they do not have a known electron transfer function. As a result, proteins could serve not only as sensing, polar,or photoactive elements in devices (such as field-effect transistor configurations) but also as electronic conductors. Current knowledge of peptide synthesis and protein modification paves the way toward a greater understanding of how changes in a protein's structure affect its conductivity. Such an approach could minimize the need for biochemical cascades in systems such as enzyme-based circuits, which transduce the protein's response to electronic current. In addition, as precision and sensitivity of solid-state measurements increase, and as knowledge of the structure and function of "dry" proteins grows, electronic conductivity may become an additional approach to study electron transfer in proteins and solvent effects without the introduction of donor or acceptor moieties. We are particularly interested in whether evolution might have prompted the electronic carrier transport capabilities of proteins for which no electrically active function is known in their native biological environment and anticipate that further research may help address this fascinating question.

Seeing the light: A joint experimental and theoretical approach delivers the free gasphase high-resolution absorption spectrum of the chromophore conformations implicated In the spectroscopy of retinal proteins (see scheme). Notably, many retinal pigments absorb close to the gasphase value. Chemical equation presentation

Electron transfer (ET) through proteins, a fundamental element of many biochemical reactions, is studied Intensively in aqueous solutions. Over the past decade, attempts were made to Integrate proteins into solid-state Junctions in order to study their electronic conductance properties. Most such studies to date were conducted with one or very few molecules In the Junction, using scanning probe techniques. Here we present the high-yield, reproducible preparation of large-area monolayer Junctions, assembled on a Si platform, of proteins of three different families: azurln (Az), a blue-copper ET protein, bacterlorhodopsln (bR), a membrane proteln-chromophore complex with a proton pumping function, and bovine serum albumin (BSA). We achieve highly reproducible electrical current measurements with these three types of monolayers using appropriate top electrodes. Notably, the current-voltage (i-V) measurements on such Junctions show relatively minor differences between Az and bR, even though the latter lacks any known ET function. Electron Transport (ETp) across both Az and bR is much more efficient than across BSA, but even for the latter the measured currents are higher than those through a monolayer of organic, C18 alkyl chains that is about half as wide, therefore suggesting transport mechanlsm(s) different from the often considered coherent mechanism. Our results show that the employed proteins maintain their conformation under these conditions. The relatively efficient ETp through these proteins opens up possibilities for using such blomolecules as current-carrying elements in solid-state electronic devices.

Structural restraints provided by solid-state NMR measurements of the metarhodopsin 11 intermediate are combined with molecular dynamics simulations to help visualize structural changes in the light activation of rhodopsin. Since the timescale for the formation of the metarhodopsin H intermediate (>1 ms) is beyond that readily accessible by molecular dynamics, we use NMR distance restraints derived from (13)C dipolar recoupling measurements to guide the simulations. The simulations yield a working model for how photoisomerization of the 11-cis retinylidene chromophore bound within the interior of rhodopsin is coupled to transmembrane helix motion and receptor activation. The mechanism of activation that emerges is that multiple switches on the extracellular (or intradiscal) side of rhodopsin trigger structural changes that converge to disrupt the ionic lock between helices H3 and H6 on the intracellular side of the receptor. (C) 2009 Elsevier Ltd. All rights reserved.

Internal conversion and energy transfer from S₂ of the salinixanthine antenna in xanthorhodopsin takes less than 30 fs, leading to lower singlets with rotated transition dipoles. This timescale questions models of resonant electronic energy transfer.

Halorhodopsin from Natronomonas pharaonis (NpHR) is a member of the retinal protein group and serves as a light-driven chloride pump in which chloride ions are transported through the membrane following light absorption by the retinal chromophore. In this study, we examined two main issues: (1) factors controlling the binding of the retinal chromophore to the NpHR opsin and (2) the ability of the NpHR opsin to catalyze the thermal isomerization of retinal isomers. We have revealed that the reconstitution process of pharaonis HR (NpHR) pigment from its apoprotein and all-trans retinal depends on the pH, and the process has a pK(a) of 5.8 +/- 0.1. It was proposed that this pK(a) is associated with the pK(a) of the lysine residue that binds the retinal chromophore (Lys256). The pigment formation is regulated by the concentration of sodium chloride, and the maximum yield was observed at 3.7 M NaCl. The low yield of pigment in a lower concentration of NaCl (

The visual pigment rhodopsin is unique among the G protein-coupled receptors in having an 11-cis retinal chromophore covalently bound to the protein through a protonated Schiff base linkage. The chromophore locks the visual receptor in an inactive conformation through specific steric and electrostatic interactions. This efficient inverse agonist is rapidly converted to an agonist, the unprotonated Schiff base of all-trans retinal, upon light activation. Here, we use magic angle spinning NMR spectroscopy to obtain the ¹³C chemical shifts (C5-C20) of the all-trans retinylidene chromophore and the ¹⁵N chemical shift of the Schiff base nitrogen in the active metarhodopsin II intermediate. The retinal chemical shifts are sensitive to the conformation of the chromophore and its molecular interactions within the protein-binding site. Comparison of the retinal chemical shifts in metarhodopsin II with those of retinal model compounds reveals that the Schiff base environment is polar. In particular, the ¹³C15 and ¹⁵Nε chemical shifts indicate that the CdN bond is highly polarized in a manner that would facilitate Schiff base hydrolysis. We show that a strong perturbation of the retinal ¹³C12 chemical shift observed in rhodopsin is reduced in wild-type metarhodopsin II and in the E181Q mutant of rhodopsin. On the basis of the T₁ relaxation time of the retinal ¹³C18 methyl group and the conjugated retinal ¹³C5 and ¹³C8 chemical shifts, we have determined that the conformation of the retinal C6-C7 single bond connecting the β-ionone ring and the retinylidene chain is 6-s-cis in both the inactive and the active states of rhodopsin. These results are discussed within the general framework of ligand-activated G protein-coupled receptors.

Two and three pulses experiments are conducted to record the elusive S1 vibrational spectrum of Retinal Protonated Schiff-Base in solution. We find a reduction in C=C stretching frequency and other shifted bands in the fluorescent state, whose relevance are discussed.

Rhodopsin is a highly specialized G protein-coupled receptor (GPCR) that is activated by the rapid photochemical isomerization of its covalently bound 11-cis-retinal chromophore. Using two-dimensional solid-state NMR spectroscopy, we defined the position of the retinal in the active metarhodopsin II intermediate. Distance constraints were obtained between amino acids in the retinal binding site and specific ¹³C-labeled sites located on the β-ionone ring, polyene chain, and Schiff base end of the retinal. We show that the retinal C20 methyl group rotates toward the second extracellular loop (EL2), which forms a cap on the retinal binding site in the inactive receptor. Despite the trajectory of the methyl group, we observed an increase in the C20-Gly¹⁸⁸ (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the β-ionone ring moves to a position between Met²⁰⁷ and Phe²⁰⁸ on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the C∈ carbons of Lys²⁹⁶ (H7) and Met⁴⁴ (H1) and between Gly¹²¹ (H3) and the retinal C18 methyl group. Helix-helix interactions involving the H3-H5 and H4-H5 interfaces were also found to change in the formation of metarhodopsin II reflecting increased retinal-protein interactions in the region of Glu¹²² (H3) and His²¹¹ (H5). We discuss the location of the retinal in metarhodopsin II and its interaction with sequence motifs, which are highly conserved across the pharmaceutically important class A GPCR family, with respect to the mechanism of receptor activation.

Controlling the orientation of bacteriorhodopsin (bR) monolayers is an important step in studying and utilizing such membranes in a solid-state configuration in., for example, photoelectric applications. Macroscopic monolayers of bR have been fabricated in a variety of ways, but characterization of the distribution of the two possible orientations in which the membrane fragments can adsorb has not yet been addressed experimentally. Here, an approach is presented that labels only one of the membrane surfaces by electroless growth of metal nanoparticles on top of the solid-supported membranes. In, this way, it is possible to observe which surface of the membranes is actually adsorbed to the substrate. How this technique serves to interface the membranes with a top metal contact for further electrical measurements is also demonstrated.

The interfacing of functional proteins with solid supports and the study of related protein-adsorption behavior are promising and important for potential device applications. In this study, we describe the preparation of bacteriorhodopsin (bR) monolayers on Br-terminated solid supports through covalent attachment. The bonding, by chemical reaction of the exposed free amine groups of bR with the pendant Br group of the chemically modified solid surface, was confirmed both by negative AFM results obtained when acetylated bR (instead of native bR) was used as a control and by weak bands observed at around 1610 cm^-1 in the FTIR spectrum. The coverage of the resultant bR monolayer was significantly increased by changing the pH of the purple-membrane suspension from 9.2 to 6.8. Although bR, which is an exceptionally stable protein, showed a pronounced loss of its photoactivity in these bR monolayers, it retained full photoactivity after covalent binding to Br-terminated alkyls in solution. Several characterization methods, including atomic force microscopy (AFM), contact potential difference (CPD) measurements, and UV/Vis and Fourier transform infrared (FTIR) spectroscopy, verified that these bR monolayers behaved significantly different from native bR. Current-voltage (I-V) measurements (and optical absorption spectroscopy) suggest that the retinal chromophore is probably still present in the protein, whereas the UV/Vis spectrum suggests that it lacks the characteristic covalent protonated Schiff base linkage. This finding sheds light on the unique interactions of biomolecules with solid surfaces and may be significant for the design of protein-containing device structures.

Molecular electronics is very much about contacts, and thus understanding of any generic contact effect is essential to its advance. For example, it is still not obvious in how far variations in electrode roughness of macroscopic contacts can lead to rectification. Here we report an investigation of this contact effect on electronic transport properties using metal-insulator-metal planar junctions with a 5 nm thick bacteriorhodopsin-based insulator as model system. We demonstrate that the experimentally observed rectifying behavior is not an intrinsic property of the molecules used, but rather of the local contact quality. Even a slight increase in surface roughness of the bottom electrode gives rise to distinct rectifying behavior in these and, by extrapolation, possibly other molecular junctions.

Incorporation of photodynamic therapy into clinical practice for induction of vascular photo-occlusion highlights the need to prevent adverse phototoxicity to sensitive juxtaposed tissues, particularly in the retina. We developed a system termed "competitive quenching" to prevent adverse phototoxic damage. It involves differential compartmentalization of a photoactivator to the intravascular compartment for photoexcitation and delivery of phototoxicity to targeted vessels. A different photodynamic agent is partitioned to the extravascular retinal space to quench reactive oxygen species generated by photosensitization, thereby protecting the adjacent retinal tissues from adverse phototoxicity. The absorption spectra of quenchers must span wavelengths that are shorter and excluded from the spectral range of photoexcitation light to prevent photoactivation of the quencher. Perihydroxylated perylenequinones were found to be suitable to function as "competitive quenchers" with the prototype hypericin identified as a potent quencher. Here we examined the mechanisms operative in competitive quenching and suggest that hypericin forms a complex with verteporfin, thereby quenching singlet oxygen formation. Furthermore, we show that hypericin, with six phenolic hydroxyls, protects retinal and endothelial hybridoma cells from phototoxicity more effectively than the dimethyl tetrahydroxy helianthrone structural analog with only four such phenolic hydroxyls. The findings suggest that hydroxyl numbers contribute to the efficacy of competitive quenching.

A retinal Schiff base analogue which artificially mimics the protein-induced red shifting of absorption in bacteriorhodopsin (BR) has been investigated with femtosecond multichannel pump probe spectroscopy. The objective is to determine if the catalysis of retinal internal conversion in the native protein BR, which absorbs at 570 nm, is directly correlated with the protein-induced Stokes shifting of this absorption band otherwise known as the "opsin shift". Results demonstrate that the red shift afforded in the model system does not hasten internal conversion relative to that taking place in a free retinal-protonated Schiff base (RPSB) in methanol solution, and stimulated emission takes place with biexponential kinetics and characteristic timescales of approximately 2 and 10.5 ps. This shows that interactions between the prosthetic group and the protein that lead to the opsin shift in BR are not directly involved in reducing the excited-state lifetime by nearly an order of magnitude. A sub-picosecond phase of spectral evolution, analogues of which are detected in photoexcited retinal proteins and RPSBs in solution, is observed after excitation anywhere within the intense visible absorption band. It consists of a large and discontinuous spectral shift in excited-state absorption and is assigned to electronic relaxation between excited states, a scenario which might also be relevant to those systems as well. Finally, a transient excess bleach component that tunes with the excitation wavelength is detected in the data and tentatively assigned to inhomogeneous broadening in the ground state absorption band. Possible sources of such inhomogeneity and its relevance to native RPSB photochemistry are discussed.

Sub-10-fs laser pulses are used to impulsively photoexcite bacteriorhodopsin (BR) suspensions and probe the evolution of the resulting vibrational wave packets. Fourier analysis of the spectral modulations induced by transform-limited as well as linearly chirped excitation pulses allows the delineation of excited- and ground-state contributions to the data. On the basis of amplitude and phase variations of the modulations as a function of the dispersed probe wavelength, periodic modulations in absorption above 540 nm are assigned to ground-state vibrational coherences induced by resonance impulsive Raman spectral activity (RISRS). Probing at wavelengths below 540 nm-the red edge of the intense excited-state absorption band-uncovers new vibrational features which are accordingly assigned to wave packet motions along bound coordinates on the short-lived reactive electronic surface. They consist of high- and low-frequency shoulders adjacent to the strong C=C stretching and methyl rock modes, respectively, which have ground-state frequencies of 1008 and 1530 cm^-1. Brief activity centered at ∼900 cm^-1, which is characteristic of ground-state HOOP modes, and strong modulations in the torsional frequency range appear as well. Possible assignments of the bands and their implication to photoinduced reaction dynamics in BR are discussed. Reasons for the absence of similar signatures in the pump-probe spectral modulations at longer probing wavelengths are considered as well.

Halorhodopsin from Natronobacterium pharaonis (pHR) is a light-driven chloride pump in which photoisomerzation of a retinal chromophore triggers a photocycle which leads to a chloride anion transport across the plasma membrane. Similarly to other retinal proteins the protonated Schiff base (PSB), which covalently links the retinal to the protein, does not experience hydrolysis reaction at room temperature even though several water molecules are located in the protonated Schiff base (PSB) vicinity. In the present studies we have revealed that in contrast to other studied archaeal rhodopsins, temperature increase to about 70°C hydrolyses the PSB linkage of pHR. The rate of the reaction is affected by Cl^- concentration and reveals an anion binding site (in addition to the Cl^- in the SB vicinity) with a binding constant of 100mM (measured at 70°C). We suggest that this binding site is located on the extracellular side and its possible role in the Cl ^- pumping mechanism is discussed. The rate of the hydrolysis reaction is affected by the nature of the anion bound to pHR. Substitution of the Cl^- anion by Br^-, I^- and SCN^- exhibits similar behavior to that of Cl^- in the region of 100mM but higher concentrations are needed for N₃^-, HCOO^- and NO₂^- to achieve similar behavior. Steady state pigment illumination accelerates the reaction and reduces the energy of activation and the frequency factor. Adjusting the sample temperature to 25°C following the hydrolysis reaction led to about 80% pigment recovery. However, the newly reformed pigment is different from the mother pigment and has different characteristics. It is concluded that the apo-membrane adopts a modified conformation and/or aggregated state which rebinds the retinal to give a new conformation of the pHR pigment.

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm^-1 for the K state of 13C-BR (13C-BR_K) versus a vibration at 957 cm^-1 for the K state of AT-BR (AT-BR _K). In AT-BR_K, but not in 13C-BR_K, the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR_K were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BRx. These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR_K, it is located elsewhere in 13C-BR_K. By use of [ζ-¹⁵N]lysine- labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D₂O) at 2173 and 2056 cm^-1, close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm^-1 in 13C-BR_K versus values of 2495 and 2468 cm^-1 for AT-BR_K, suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR_K does not occur in 13C-BR_K. Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.

The visual pigment rhodopsin is a seven-transmembrane (7-TM) G protein-coupled receptor (GPCR). Activation of rhodopsin involves two pH-dependent steps: proton uptake at a conserved cytoplasmic motif between TM helices 3 and 6, and disruption of a salt bridge between a protonated Schiff base (PSB) and its carboxylate counterion in the transmembrane core of the receptor. Formation of an artificial pigment with a retinal chromophore fluorinated at C14 decreases the intrinsic pK_a of the PSB and thereby destabilizes this salt bridge. Using Fourier transform infrared difference and UV-visible spectroscopy, we characterized the pH-dependent equilibrium between the active photoproduct Meta II and its inactive precursor, Meta I, in the 14-fluoro (14-F) analogue pigment. The 14-F chromophore decreases the enthalpy change of the Meta I-to-Meta II transition and shifts the Meta I/Meta II equilibrium toward Meta II. Combining C14 fluorination with deletion of the retinal β-ionone ring to form a 14-F acyclic artificial pigment uncouples disruption of the Schiff base salt bridge from transition to Meta II and in particular from the cytoplasmic proton uptake reaction, as confirmed by combining the 14-F acyclic chromophore with the E134Q mutant. The 14-F acyclic analogue formed a stable Meta I state with a deprotonated Schiff base and an at least partially protonated protein counterion. The combination of retinal modification and site-directed mutagenesis reveals that disruption of the protonated Schiff base salt bridge is the most important step thermodynamically in the transition from Meta I to Meta II. This finding is particularly important since deprotonation of the retinal PSB is known to precede the transition to the active state in rhodopsin activation and is consistent with models of agonist-dependent activation of other GPCRs.

Halorhodopsin from Natronomonas pharaonis (pHR) is a light-driven chloride pump that transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. Analysis of the amino acid sequence of pHR reveals three cysteine residues (Cys160, Cys184, and Cys186) in helices D and E. Here we have labeled the cysteine residues with nitroxide spin labels and studied using electron paramagnetic resonance (EPR) spectroscopy their mobility, accessibility to various reagents, and the distance between the labels. It was revealed by following the d₁/d parameter that the distance between the spin labels is ca. 13-15 Å. The EPR spectrum suggests that one label has a restricted mobility while the other two are more mobile. Only one label is accessible to hydrophilic paramagnetic broadening reagents leading to the conclusion that this label is exposed to the water phase. All three labels are reduced by ascorbic acid and reoxidized by molecular oxygen. The rate of the oxidation is accelerated following retinal irradiation indicating that the protein experiences conformation alterations in the vicinity of the labels during the pigment photocycle. It is suggested that Cys186 is exposed to the bulk medium while Cys184, located close to the retinal ionone ring, exhibits an immobilized EPR signal and is characterized by a hydrophobic environment.

A visible-pump/UV-probe transient absorption is used to characterize the ultrafast dynamics of bacteriorhodopsin with 80-fs time resolution. We identify three spectral components in the 265- to 310-nm region, related to the all-trans retinal, tryptophan (Trp)-86 and the isomerized photoproduct, allowing us to map the dynamics from reactants to products, along with the response of Trp amino acids. The signal of the photoproduct appears with a time delay of ≈250 fs and is characterized by a steep rise (≈150 fs), followed by additional rise and decay components, with time scales characteristic of the J intermediate. The delayed onset and the steep rise point to an impulsive formation of a transition state on the way to isomerization. We argue that this impulsive formation results from a splitting of a wave packet of torsional modes on the potential surface at the branching between the all-trans and the cis forms. Parallel to these dynamics, the signal caused by Trp response rises in ≈200 fs, because of the translocation of charge along the conjugate chain, and possible mechanisms are presented, which trigger isomerization.

Photochemistry of retinal protonated Schiff-base analogue mimicking opsin shift of bacteriorhodopsin is studied. Results rule out correlations between BR's absorption red shifting and internal conversion catalysis. Indications for the involvement of multiple excited states uncovered.

Acetylation of purple membranes ( PM) significantly enhances the surface photovoltage that they exhibit, if adsorbed as a monolayer on a solid surface; we suggest that this increase is due to the improved orientation of the PM on the surface.

Sensory rhodopsin II, a repellent phototaxis receptor from Natronomonas (Natronobacterium) pharaonis (NpSRII), forms a complex with its cognate transducer (NpHtrII). In micelles the two proteins form a 1:1 heterodimer, whereas in membranes they assemble to a 2:2 complex. Similarly to other retinal proteins, sensory rhodopsin II undergoes a bleaching reaction with hydroxylamine in the dark which is markedly catalyzed by light. The reaction involves cleavage of the protonated Schiff base bond which covalently connects the retinal chromophore to the protein. The light acceleration reflects protein conformation alterations, at least in the retinal binding site, and thus allows for detection of these changes in various conditions. In this work we have followed the hydroxylamine reaction at different temperatures with and without the cognate transducer. We have found that light irradiation reduces the activation energy of the hydroxylamine reaction as well as the frequency factor. A similar effect was found previously for bacteriorhodopsin. The interaction with the transducer altered the light effect both in detergent and membranes. The transducer interaction decreased the apparent light effect on the energy of activation and the frequency factor in detergent but increased it in membranes. In addition, we have employed an artificial pigment derived from a retinal analog in which the critical C₁₃=C₁₄ double bond is locked by a rigid ring structure preventing its isomerization. We have observed light enhancement of the reaction rate and reduction of the energy of activation as well as the frequency factor, despite the fact that this pigment does not experience C₁₃=C₁₄ double bond isomerization. It is suggested that retinal excited state polarization caused by light absorption of the "locked" pigment polarizes the protein and triggers relatively long-lived protein conformational alterations.

The retinal protein protonated Schiff base linkage plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment, the Schiff base (SB) is titrated with a pK_a of ∼13, but following light absorption, it experiences a decrease in the pK_a and undergoes several alterations, including a deprotonation process. We have studied the SB titration using retinal analogues which have intrinsically lower pK_a's which allow for SB titrations over a much lower pH range. We found that above pH 9 the channel for the SB titration is perturbed, and the titration rate is considerably reduced. On the basis of studies with several mutants, it is suggested that the protonation state of residue Glu204 is responsible for the channel perturbation. We suggest that above pH 12 a channel for the SB titration is restored probably due to titration of an additional protein residue. The observations may imply that during the bR photocycle and M photointermediate formation the rate of Schiff base protonation from the bulk is decreased. This rate decrease may be due to the deprotonation process of the "proton-releasing complex" which includes Glu204. In contrast, during the lifetime of the O intermediate, the protonated SB is exposed to the bulk. Possible implications for the switch mechanism, and the directionality of the proton movement, are discussed.

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal micro-crystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum ²H NMR as well as ¹H and ²H diffusion NMR to characterize the interaction of water molecules with the PM in D₂O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (υ_q ∼5.5 Hz, 298 K, at 11.7 T) is observed for the D ₂O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (τ_max), and the relative intensity of the ²H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D₂O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D₂O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D₂O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D₂O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, ¹H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.

Activation of the visual pigment rhodopsin is caused by 11-cis to -trans isomerization of its retinal chromophore. High-resolution solid-state NMR measurements on both rhodopsin and the metarhodopsin II intermediate show how retinal isomerization disrupts helix interactions that lock the receptor off in the dark. We made 2D dipolar-assisted rotational resonance NMR measurements between ¹³C-labels on the retinal chromophore and specific ¹³C-labels on tyrosine, glycine, serine, and threonine in the retinal binding site of rhodopsin. The essential aspects of the isomerization trajectory are a large rotation of the C20 methyl group toward extracellular loop 2 and a 4- to 5-Å translation of the retinal chromophore toward transmembrane helix 5. The retinal-protein contacts observed in the active metarhodopsin II intermediate suggest a general activation mechanism for class A G protein-coupled receptors involving coupled motion of transmembrane helices 5, 6, and 7.

Two dimensional (2D) solid-state ¹³C...¹³C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B₁ fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish ¹³C-¹³C correlations in rhodopsin. In rhodopsin containing 4-¹³C-Tyr and 8,19-¹³C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-¹³CH₃, which are 4.8 Å apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-¹³CH₃, which are 5.5 Å apart in the crystal structure. These data demonstrate that long range ¹³C...¹³C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-¹³C Gly121 and U-¹³C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 Å) in the crystal structure. Based on model compounds, the absence of a ¹³C...¹³C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.

Low frequency excited state vibrational coherences induced by impulsive photoexcitation in bacteriorhodopsin are detected via femtosecond pump-probe spectroscopy, and compared with similar data in retinal protonated Schiff bases of native and locked retinals. At delays above ∼100 fs a single vibration below 200 fs dominates the detected spectral modulations. Its frequency of ∼120 in retinal protonated Schiff base is virtually unchanged by locking the C₁₃=C₁₄ bond in the trans or cis configurations, but is increased to 170 cm^-1 within the protein environment. The implications of this result on the part played by the protein in directing the reactivity of the retinal within bacteriorhodopsin is discussed.

Light-induced isomerization of rhodopsin's retinal chromophore to the activating all-trans geometry initializes the formation of the active receptor state, Meta II. In the absence of peripheral regulatory proteins, the activity of Meta II is switched off spontaneously by two independent pathways: either by hydrolysis of the retinal Schiff base and dissociation of the light receptor into apoprotein opsin plus free retinal or by formation of Meta III, an inactive species with intact retinal protonated Schiff base absorbing at 470 nm. By FTIR spectroscopy on rhodopsin reconstituted with isotopically labeled chromophores in combination with quantum mechanical DFT calculations, we show that the deactivating step during formation of Meta III involves a thermal isomerization of the chromophore C=N double bond, such that the chromophore in Meta III is all-trans-15-syn. This isomerization step is catalyzed by the protein environment and proceeds via Meta I, as suggested by its dependence on pH and on properties of the lipid/detergent environment of the protein. In the long term, Meta III decays likewise to opsin and free retinal by slow hydrolysis of the Schiff base.

The visual pigment rhodopsin is characterized by an 11-cis retinal chromophore bound to Lys-296 via a protonated Schiff base. Following light absorption the C₁₁=C₁₂ double bond isomerizes to trans configuration and triggers protein conformational alterations. These alterations lead to the formation of an active intermediate (Meta II), which binds and activates the visual G protein, transducin. We have examined by UV-visible and Fourier transform IR spectroscopy the photochemistry of a rhodopsin analogue with an 11-cis-locked chromophore, where cis to trans isomerization around the C₁₁=C₁₂ double bond is prevented by a 6-member ring structure (Rh_6.10). Despite this lock, the pigment was found capable of forming an active photoproduct with a characteristic protein conformation similar to that of native Meta II. This intermediate is further characterized by a protonated Schiff base and protonated Glu-113, as well as by its ability to bind a transducin-derived peptide previously shown to interact efficiently with native Meta II. The yield of this active photointermediate is pH-dependent and decreases with increasing pH. This study shows that with the C₁₁=C₁₂ double bond being locked, isomerization around the C₉=C₁₀ or the C₁₃=C₁₄ double bonds may well lead to an activation of the receptor. Additionally, prolonged illumination at pH 7.5 produces a new photoproduct absorbing at 385 nm, which, however, does not exhibit the characteristic active protein conformation.

Bacteriorhodopsin's photocycle is initiated by the retinal chromophore light absorption. It has usually been assumed that light primarily isomerizes a retinal double bond which in turn induces protein conformational alterations and biological activity. We have studied several artificial pigments derived from retinal analogues tailored to substantially reduce the light-induced chromophore polarization. The lack of chromophore polarization was reflected in an undetectable second harmonic generation (SHG) signal. It was revealed that these artificial pigments did not exhibit any detectable light-induced photocycle nor light acceleration of the hydroxylamine-bleaching reaction. We suggest that light-induced retinal polarization triggers protein polarization which controls the course of the isomerization reaction by determining the relative efficiency of forward versus back-branching processes.

The retinal protonated Schiff base of bacteriorhodopsin is photoreactive to reducing agents such as NaBH₄. In the present work we have studied the effect of different protein hydration levels on the photoreductive reaction, as well as the consequences of preventing isomerization around the critical C₁₃=C₁₄ retinal double bond. It was revealed that the rate of light-induced NaBH₄ reaction can be fitted to three phases, between 100 and 87%, from 87 to 35% and below 35% relative humidities (r.h.). The three phases are attributed to three protein regions characterized by different water affinities. Furthermore, it is shown that the PSB reduction reaction is light catalyzed even in artificial pigments derived from retinal analogs, in which isomerization around the C₁₃=C₁₄ double bond is prevented. It is suggested that the protein experiences light-induced conformational alterations that are not associated with C₁₃=C₁₄ double bond isomerization. In the 13-cis locked pigment the rate of reduction reaction is affected by r.h. levels only below 35%. The relatively low r.h. required for withdrawing water from the protein is attributed to the increased protein-water affinity in this specific pigment.

The molecular mechanism describing the initial 200 ps of the room-temperature photocycle in the artificial bacteriorhodopsin (BR) pigment, BR6.9, is examined by both absorption and vibrational spectroscopy. The BR6.9 pigment contains a structurally modified retinal chromophore (retinal 6.9) having a six-membered carbon ring bridging the C₉=C₁₀-C₁₁ bonds. Picosecond transient absorption (PTA) data show that the initial 200-ps interval of the BR6.9 photocycle contains two intermediates: J6.9 formed with a 5-ns lifetime). Resonantly enhanced vibrational spectra from the light- and dark-adapted ground states of BR6.9 are measured using picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). Each of these PR/CARS spectra (800-1700 cm^-1) contains 33 features assignable to the vibrational degrees of freedom in the retinal chromophore. CARS spectra from the K6.9, obtained from picosecond time-resolved CARS (PTR/CARS) data using 10-ps, 50-ps, 100-ps, and 200-ps time delays following the 570-nm initiation of the BR6.9 photocycle, contain a comparable number of features assignable to the retinal in K6.9. The vibrational spectrum of J6.9 can be tentatively characterized by two bands observed in the 1120-1200-cm^-1 region from the analysis of the 10-ps PTR/CARS data. Comparisons involving these PTA and CARS data from BR6.9, as well as analogous results obtained from the ground states and photocycle intermediates of native BR and other artificial BR pigments, demonstrate that restricting retinal motion at the C₉=C₁₀-C₁₁ bonds does not generally change the initial 200-ps photocycle mechanism, but does alter the rates at which specific molecular processes occur. These vibrational CARS spectra show that the retinal structures in K6.9 and in both light- and dark-adapted BR6.9 are all distinct. However, the specific mechanistic role, if any, of C₁₃=C₁₄ isomerization cannot be directly identified from CARS data recorded from BR6.9 and its photocycle intermediates. Even though C₁₃=C₁₄ isomerization has been widely considered the primary retinal structural change underlying the proton-pumping mechanism in BR pigments, these results leave open the question of whether C₁₃=C₁₄ isomerization is required as a mechanistic precursor for biochemical activity in BR pigments such as BR6.9.

Femtosecond stimulated emission pumping experiments from bacteriorhodopsins fluorescent state show for the first time that it is a photocycle intermediate, with a constant nonisomerized structure throughout its 0.5 psec lifetime. Implications concerning internal conversion dynamics are discussed.

Time-integrated fluorescence experiments on native bacteriorhodopsin and on its non-isomerizing form bR5.12 are reported. The experimental set-up was designed such as to observe emission exclusively from the excited state intermediate I-460. We obtain the first systematic investigation of the fluorescence spectra as a function of the excitation wavelength tuned throughout the entire absorption band of bR. An important finding is that the position of the fluorescence maximum does not show a systematic shift when the excitation wavelength is shortened. For excitation with high excess energy, we observe a broadening of the blue wing of the bR fluorescence, indicating incomplete vibrational energy relaxation on the time scale of the lifetime of I-460. Due to a much longer excited state lifetime, vibrational energy relaxation is more e3ective in bR5.12 and the fluorescence spectra are much less dependent on excitation wavelength. The results are placed in the general framework of thermalization between the retinal chromophore and the protein environment, and are compared with information obtained by femtosecond experiments.

The primary events in the photosynthetic retinal protein bacteriorhodopsin (bR) are reviewed in light of photophysical and photochemical experiments with artificial bR in which the native retinal polyene is replaced by a variety of chromophores. Focus is on retinals in which the "critical" C₁₃=C₁₄ bond is locked with respect to isomerization by a rigid ring structure. Other systems include retinal oxime and non-isomerizable dyes noncovalently residing in the binding site. The early photophysical events are analyzed in view of recent pump-probe experiments with sub-picosecond time resolution comparing the behavior of bR pigments with those of model protonated Schiff bases in solution. An additional approach is based on the light-induced cleavage of the protonated Schiff base bond that links retinal to the protein by reacting with hydroxylamine. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that in bR the initial relaxation out of the Franck-Condon (FC) state does not involve substantial C₁₃=C₁₄ torsional motion and is considerably catalyzed by the protein matrix. Prior to the decay of the relaxed fluorescent state (FS or I state), the protein is activated via a mechanism that does not require double bond isomerization. Most plausibly, it is a result of charge delocalization in the excited state of the polyene (or other) chromophores. More generally, it is concluded that proteins and other macromolecules may undergo structural changes (that may affect their chemical reactivity) following optical excitation of an appropriately (covalently or non-covalently) bound chromophore. Possible relations between the light-induced changes due to charge delocalization, and those associated with C₁₃=C₁₄ isomerization (that are at the basis of the bR photocycle), are discussed. It is suggested that the two effects may couple at a certain stage of the photocycle, and it is the combination of the two that drives the cross-membrane proton pump mechanism.

Absorbance difference spectra were recorded from 20 ns to 1 mus after 20 T photoexcitation of artificial visual pigments derived either from 5-demethylretinal or from a mesityl analogue of retinal. Both pigments produced an early photointermediate similar to bovine bathorhodopsin (Batho). In both cases the Batho analogue decayed to a lumirhodopsin (Lumi) analogue via a blue-shifted intermediate, BSI, which formed an equilibrium with the Batho analogue. The stability of 5-demethyl Batho, even though the C8-hydrogen of the polyene chain cannot interact with a ring C5-methyl group to provide a barrier to Batho decay, raises the possibility that the 5-demethylretinal ring binds oppositely from normal to form a pigment with a 6-s-trans ring-chain conformation. If 6-s-trans binding occurred, the ring Cl-methyls could replace the C5-methyl in its interaction with the chain C8-hydrogen to preserve the steric barrier to Batho decay, consistent with the kinetic results. The possibility of 6-s-trans binding for 5-demethylretinal also could account for the unexpected blue shift of 5-demethyl visual pigments and could explain why 5-demethyl artificial pigments regenerate so slowly. Although the mesityl analogue BSI's absorption spectrum was blue-shifted relative to its pigment spectrum, the blue shift was much smaller than for rhodopsin's or 5-demethylisorhodopsin's BSI. This suggests that increased C6-C7 torsion may be responsible for some of BSI's blue shift, which is not the case for mesityl analogue BSI either because of reduced spectral sensitivity to C6-C7 torsion or because the symmetry of the mesityl retinal analogue precludes having 6-s-cis and 6-s-trans conformers. The similarity of the mesityl analogue BSI and native BSI pi (max) values supports the idea that BSI has a 6-s angle near 90 degrees, a condition which could disconnect the chain (and BSI's spectrum) from the double bond specifics of the ring.

We studied the salt dependence of both the stability and the equilibrium of the late photoproducts metarhodopsin I (MI) and II (MII) of the artificial visual pigment 9-demethyl rhodopsin (9dm-Rho). In the photoproducts of 9dm-Rho, all-trans-9-demethyl retinal acts only as a partial agonist, enabling us to study the photoproduct equilibrium of the pigment both in membranes and in detergent micelles. Chloride, bromide, and phosphate salts shift this equilibrium from the inactive MI to the active MII receptor conformation both in native membranes and even more with purified pigment in detergent micelles. In the presence of these salts, the induced MII state seems to be structurally intact, as judged by Fourier transform infrared (FTIR) and UV - vis spectroscopy. In the long term, however, we observe an increased instability of the photoproducts and a change in the decay pathways. Both MII enhancement and destabilization are particularly pronounced with the strong chaotropic salts KI and KSCN. The results fit into the framework of the Hofmeister effect and are assigned to an increased solvation of the peptide moiety of the solvent-exposed domains, their resulting partial disordering favoring MII over MI. In this picture, increased solvation also affects helix - helix interactions, thereby leading to a structural instability of the protein in the long term. The reported influences of salts on conformation and stability of this membrane protein are likely to be general and may therefore also apply to other transmembrane proteins and particularly to other G protein-coupled receptors.

The photoactivation of retinal proteins is usually interpreted in terms of C=C photoisomerization of the retinal moiety, which triggers appropriate conformational changes in the protein. In this work several dye molecules, characterized by a completely rigid structure in which no double-bond isomerization is possible, were incorporated into the binding site of bacteriorhodopsin (bR). Using a light-induced chemical reaction of a labeled EPR probe, it was observed that specific conformational alterations in the protein are induced following light absorption by the dye molecules occupying the binding site. The exact nature of these changes and their relationship to those occurring in the bR photocycle are still unclear. Nevertheless, their occurrence proves that C=C or C=NH⁺ isomerization is not a prerequisite for protein conformational changes in a retinal protein. More generally, we show that conformational changes, leading to changes in reactivity, may be induced in proteins by optical excitation of simple nonisomerizable dyes located in the macromolecular matrix.

The mechanism by which bacteriorhodopsin is activated following light absorption is not completely clear. We have detected protein conformational alterations following light absorption by retinal-based chromophores in the bacteriorhodopsin binding site by monitoring the rate of reduction-oxidation reactions of covalently attached spin labels, using EPR spectroscopy. It was found that the reduction reaction with hydrolxylamine is light-catalyzed in the A103C-labeled pigment but not in E74C or M163C. The reaction is light- catalyzed even when isomerization of the C₁₃=C₁₄ bond of the retinal chromophore is prevented. The reverse oxidation reaction with molecular oxygen is effective only in apomembrane derived from the mutant A103C. This reaction is light-accelerated following light absorption of the retinal oxime, which occupies the binding site. The light-induced acceleration is evident also in 'locked' bacteriorhodopsin in which isomerization around the C₁₃=C₁₄ bond is prevented. It is evident that the chromophore-protein covalent bond is not a prerequisite for protein response. In contrast to the case of the retinal oxime, a reduced C=N bond A103C-labeled pigment did not exhibit acceleration of the oxidation reaction following light absorption. Acceleration was observed, however, following substitution of the polyene by groups that modify the excited stated charge delocalization. It is suggested that protein conformational alterations are induced by charge redistribution along the retinal polyene following light absorption.

The effects of pH on the yield (φ(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O₆₃₀ intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and φ(r) at high pH [pK(a)(1) ~ 8]; 2) A rise in φ(r) at low pH [pK(a)(2) ~ 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) ~ 4.5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of φ(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R₁) whose state of protonation controls the magnitude of φ(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O₆₃₀ parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R₂ and R₃. R₂ affects the rise of φ(r) at low pH, whereas the state of protonation of R₃ affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.

The evolution of stimulated emission from the reactive excited state of bacteriorhodopsin is compared with that of a modified protein containing a synthetic retinal which is unable to twist around the C-13=C-14 bond. The rise of the emission is demonstrated to be structured, multistaged and wavelength dependent, appearing later in the red edge of the emission bands in both proteins. Spectral modulations associated with vibronic wavepacket motion are also observed in both. The mechanism of strong Stokes shifting of the emission resolved here, and the nature of coherences observed in the excited state are discussed. (C) 1999 Elsevier Science B.V. All rights reserved.

The primary light-induced events in the photosynthetic retinal protein bacteriorhodopsin (bR) are investigated by ultrafast optical spectroscopy over the 440-1000 nm spectral range. The study compares the early dynamics of the native all-trans pigment bR₅₇₀ with those of two synthetic analogues, bR5.12 and bR5.13, in which isomerization around the critical C₁₃=C₁₄ bond is blocked by a five-membered ring into all-trans and 13-cis configurations, respectively. Nearly identical spectral evolution is observed in both native and artificial systems over the first 100-200 fs of probe delay. During this period stimulated near-IR (∼900 nm) emission, and intense ∼460 nm absorption bands, due to analogous fluorescent I states (denoted as I₄₆₀, I5.12 and 15.13, respectively), appear concurrently within 30 fs. In all systems continuous spectral shifting over tens of femtoseconds is observed in the 500-700 nm range. Native bR goes on to produce the J₆₂₅ absorption band within ∼1 ps, which is accompanied by disappearance of the I₄₆₀ emission and absorption features. In bR5.12 and bR5.13, aside from minor spectral modifications, the analogous dramatic changes associated with I5.12 and I5.13 are arrested beyond the first ∼100 fs, reverting uniformly to the initial ground state with exponential time constants of 19 ps and 11 ps, respectively. Analysis of the data calls for a major revision of models previously put forward for the primary events in bacteriorhodopsin. The close likeness of initial transient spectral evolution in both native and artificial pigments, despite the locking of the active isomerization coordinate in the synthetic chromophores, demonstrates that in bR₅₇₀ the ultrafast changes in transmission leading to I₄₆₀, previously believed to involve C₁₃=C₁₄ torsion, must be associated with other modes. The detailed comparison conducted here also identifies which of the later spectral changes in the native system requires torsional flexibility in C₁₃=C₁₄. Similarity of 660 nm probing data in both synthetic and native chromophores demonstrates that the sub-picosecond dynamic features uncovered at this probing wavelength commonly attributed to the evolution of J₆₂₅, are not, as previously thought, reliable measures of all-trans ⇔ 13-cis isomerization dynamics.

The last stages of the photocycle of the photosynthetic pigment all- trans bacteriorhodopsin (bR₅₇₀), as well as its proton pump mechanism, are markedly pH dependent. We have measured the relative amount of the accumulated O₆₃₀ intermediate (Φ(r)), as well as its rise and decay rate constants (k(r) and k(d), respectively), over a wide pH range. The experiments were carried out in deionized membrane suspensions to which varying concentrations of metal cations and of large organic cations were added. The observed pH dependencies, s-shaped curves in the case of Φ(r) and bell-shaped curves for k(r) and k(d), are interpreted in terms of the titration of three protein residues denoted as R₁, R₂, and R₃. The R₁ titration is responsible for the increase in Φ(r), k(r), and k(d) upon lowering the pH from pH ≃ 9.5 to 7. At low pH Φ(r) exhibits a secondary rise which is attributed to the titration of a low pK(a) group, R₂. After reaching a maximum at pH ≃ 7, k(r) and k(d) undergo a decrease upon decreasing the pH, which is attributed to the titration of R₃. All three titrations exhibit pK(a) values which decrease upon increasing the salt concentration. As in the case of the Purple (bR₅₇₀) ⇆ Blue (bR₆₀₅) equilibrium, divalent cations are substantially more effective than monovalent cations in shifting the pK(a) values. Moreover, bulky organic cations are as effective as small metal cations. It is concluded that analogously to the Purple ⇆ Blue equilibrium, the salt binding sites which control the pK(a) values of R₁, R₂, and R₃ are located on, or close to, the membrane surface. Possible identifications of the three protein residues are considered. Experiments with the E204Q mutant show that the mutation has markedly affected the R₂ (Φ(r)) titration, suggesting that R₂ should be identified With Glu-204 or with a group whose pK(a) is affected by Glu-204. The relation between the R₁, R₂ and R₃ titrations and the proton pump mechanism is discussed. It is evident that the pH dependence of Φ(r) is unrelated to the measured pK(a) of the group (XH) which releases the proton to the extracellular medium during the photocycle. However, since the same residue may exhibit different pK(a) values at different stages of the photocycle, it cannot be excluded that R₂ or R₃ may be identified with XH.

The light-driven proton pump bacteriorhodopsin (bR) undergoes a bleaching reaction with hydroxylamine in the dark, which is markedly catalyzed by light. The reaction involves cleavage of the (protonated) Schiff base bond, which links the retinyl chromophore to the protein. The catalytic light effect is currently attributed to the conformational changes associated with the photocycle of all-trans bR, which is responsible for its proton pump mechanism and is initiated by the all-trans → 13-cis isomerization. This hypothesis is now being tested in a series of experiments, at various temperatures, using three artificial bR molecules in which the essential C₁₃=C₁₄ bond is locked by a rigid ring structure into an all-trans or 13- cis configuration. In all three cases we observe an enhancement of the reaction by light despite the fact that, because of locking of the C₁₃=C₁₄ bond, these molecules do not exhibit a photocycle, or any proton- pump activity. An analysis of the rate parameters excludes the possibility that the light-catalyzed reaction takes place during the ~20-ps excited state lifetimes of the locked pigments. It is concluded that the reaction is associated with a relatively long-lived (μs-ms) light-induced conformational change that is not reflected by changes in the optical spectrum of the retinyl chromophore. It is plausible that analogous changes (coupled to those of the photocycle) are also operative in the cases of native bR and visual pigments. These conclusions are discussed in view of the light-induced conformational changes recently detected in native and artificial bR with an atomic force sensor.

The experiments reported in this paper, based on reconstitution of bacteriorhodopsin (bR) from apomembrane at varying environmental conditions, demonstrate that the presence of water is a controlling factor in generating a native wild-type bR conformation. If water is lacking during this reconstitution process, then a non-native bR structure is formed that exhibits altered M formation and decay kinetics, as well as different behavior following extensive dehydration. It is shown that mutants affecting the ability of bR to form appropriate structures of water in specific protein cavities also affect the ability to generate a native bR conformation. The results suggest that aspartic acid 96 plays a major role in anchoring the appropriate water structure conformation associated with bR. It is also demonstrated that the glutamic acid 204 residue is pivotal in controlling the protein/water affinity. This water affinity can be further controlled by modifying the charge environment of the protein with altered pH. These data, based on kinetic absorption spectroscopy and Fourier transform infrared spectroscopy, highlight the central role of water in this protein.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (λ(max) = 570 nm) to the blue form (λ(max) = 605 nm) of bR (pK(a) = 3.5 in 20 mM NaCl). The Purple mutually implies Blue transition can also be induced by deionization (cation removal). These color changes offer a unique opportunity for time resolving the titration of a protein residue using conventional stopped-flow methodologies. We have studied the Purple mutually implies Blue equilibration kinetics in bR by exposing the system to pH and to cation jumps. Independently of the equilibration direction (Purple→Blue or Blue→Purple) and of the inducing concentration jump ([H⁺] or [cation]), the kinetics are found to exhibit analogous multicomponent features. Analysis of the data over a range of cation concentrations and pH values leads to the conclusion that the rate-determining step in the overall titration of Asp-85 is proton translocation through a specific proton channel. The multicomponent kinetics, extending over a wide time range (10^-2 -10⁴ s), are accounted for in terms of a pH-dependent heterogeneity of proton channels. A model is presented in which the relative weight of four proton channels is determined by the state of protonation of two interacting, channel-controlling, protein residues A₁ and A₂. These findings bear on the mechanism of the vectorial proton translocation associated with the photocycle of bR.

This paper reports on experiments that have monitored protein microsecond dynamics with a cantilevered near-field optical glass fiber. In these experiments two photoactive proteins, bacteriorhodopsin (bR) and the photosynthetic reaction center (PS I), are used to demonstrate that such probes can measure light-induced microsecond protein dynamics even though the resonance frequencies of the glass cantilevers used are in the order of a few hundred kilohertz. In the case of the light-driven proton pump, bR, the light-induced atomic force sensing (AFS) signal is negative (indicating contraction) in the microsecond time domain of the L photointermediate and becomes positive (corresponding to expansion) in the subsequent M intermediate that lives for milliseconds. Double pulse experiments from M to bR show that the latter process reverses the AFS signal. Thus, the AFS structural changes are coupled with the (optical) photocycle intermediates. Light-induced contraction and expansion phenomena are also observed in the case of PSI. In both systems the time regime of the dynamic phenomena that have been measured with AFS is five orders of magnitude faster than the fastest previously recorded atomic force detection of dynamic phenomena. This advance portends a new era in dynamic imaging of protein conformational changes.

Illumination of the Trp86 → Phe mutant of bacteriorhodopsin causes anomalous light adaptation, i.e., isomerization of the retinal from all- trans to 13-cis, 15-syn. FTIR spectral analysis shows that illumination at 250 K yields two 13-cis photoproducts, the conventional 13-cis, 15-syn state, BR(C), and another termed BR(X). BR(X) is different from BR(C) because it has a lower N-H in-plane bending frequency and a higher C₁₄-C₁₅ stretching frequency, as well as an absence of coupling between these modes. BR(X), which is stable at 275 K, is more abundant in the photosteady state produced by longer wavelength light and detected as the only photoproduct at 170 K. Its different structural features result from distortion of the C₁₄-C₁₅ bond of the chromophore. In the W86F mutant protein, the small structural changes of a water molecule in the conversion between the all-trans and 13- cis, 15-syn forms and in the formation of the K photointermediate are absent, but the larger changes of water molecule(s) that normally occur in the L and M intermediates are present. We propose that Trp86, together with Asp85, is involved in binding the water molecule and in preventing the formation of the 13-cis, 15-syn photoproducts, BR(C) and BR(X), when the wild type protein is illuminated.

The complexation between the photoreceptor sensory rhodopsin I (SRI) and its signal transducer protein HtrI was examined by assessing titration of the Schiff base chromophore of SRI with sodium hydroxide and reactivity with hydroxylamine in the presence or absence of HtrI. The apparent pK_a of the protonated Schiff base of SRI is 12.2 in the presence and 9.5 in the absence of HtrI. Direct titration of the Schiff base proton was confirmed by titrating an artificial SRI reconstituted with a 14-fluororetinal which reduces the intrinsic pK_a of the protonated Schiff base of the HtrI-complexed pigment from >12 to 9.0. The SRI chromophore exhibits high stability to hydroxylamine bleaching in the presence of HtrI; however, removal of HtrI accelerates the bleaching rate 2.4-fold. These results indicate that SRI is physically associated with HtrI in its unactivated (i.e., dark) state. In view of the previously identified association of the SRI signaling state (S₃₇₃) with HtrI, we conclude that SRI transduces the signal to HtrI through its altered interaction with the prebound transducer protein. The effect of the altered SRI/HtrI interaction on resetting the signaling state of SRI was also examined. At neutral pH the decay of S₃₇₃ is retarded by 20-fold when HtrI is absent. This effect was found to be due to a raised enthalpic barrier for the transition state during S₃₇₃ decay. The energy barrier for S₃₇₃ decay in this pigment can be lowered by providing extramembranous protons (lowering bulk pH). Therefore, light-induced alteration in SRI/HtrI interaction is important for reducing the energy barrier for S₃₇₃ decay presumably by providing or assisting a proton supply for retinal Schiff base reprotonation.

The C-C stretching vibrations (1100-1400 cm^-1) of the K-590 intermediate (containing a ¹³C_14,15 retinal), formed during the room-temperature (RT) bacteriorhodopsin (BR) photocycle, are measured using picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS). Although time-resolved resonance Raman data have been published previously for intermediates in the room-temperature BR photocycle, these PTR/ CARS data are the first time-resolved vibrational spectra from a picosecond BR intermediate at RT containing an isotopically-labeled (¹³C) retinal. The C₁₄ and C₁₅ positions are selected for isotopic labeling because motions around the C₁₃=C₁₄ and C₁₄-C₁₅ bonds are thought to underlie the structural transformation from BR-570 to K-590 and, therefore, the energy storage and transduction mechanism in the RT/BR photocycle. These PTR/CARS data are recorded 50 ps after the BR photocycle is initiated with 570-nm (5 ps, fwhm) excitation and are fit to within -1 via third-order nonlinear susceptibility (χ⁽³⁾) relationships. Comparisons of these PTR/CARS data at RT with the results from earlier resonance Raman (RR) studies of K-590 at low temperature (LT) reveal new temperature effects. Specifically, three CARS bands (1197, 1184, and 1167 cm^-1) are observed from ¹³C_14,15 K-590 in H₂O samples via PTR/CARS at RT, while only two bands (1189 and 1170 cm^-1) are found in LT/RR measurements from ¹³C_14,15 K-625. Analogous temperature-dependent differences are found in data measured from ¹³C_14,15 K-590 in D₂O samples. Independently, PTR/CARS data at RT demonstrate that deuteration of the Schiff-base nitrogen causes major changes in the fingerprint region: the 1197-cm^-1 band decreases to 1193 cm^-1 while diminishing in intensity by half and a new band appears at 1206 cm^-1. No such deuterium effect is observed in the LT/RR data from ¹³C_14,15 K-590. The previously unrecognized sensitivity of fingerprint bands to deuteration of the Schiff-base nitrogen suggests that the C-C stretching modes are highly mixed with each other and coupled to the N-H(D) retinal rocking mode. Although the temperature and Schiff-base deuteration effects reported here had not been previously identified in LT vibrational data from K-625, an analysis of the RT/CARS data continues to support the 13-cis, 14-trans retinal structure in K-590 proposed from LT results.

Vibrational spectra recorded by coherent anti-Stokes resonance Raman scattering (CARS) from bacteriorhodopsin (BR) samples containing isotopically substituted (²H and ¹³C) retinal chromophores were measured using high repetition rate, low-power, picosecond pulsed excitation (λ₁ = 580 nm and λ₈ = 640 ± 3 nm). These picosecond resonance CARS (PR/CARS) data were analyzed via third-order susceptibility relationships [χ⁽³⁾] to obtain band origins, bandwidths, relative intensities, and electronic phase factors assignable to all significant vibrational Raman features in the 1490-1700 cm^-1 wavenumber region (the ethylenic stretching and C=N-H rocking or Schiff base modes). Isotopic substitution selectively places ²H at C₁₅, ¹³C singly at the C₁₀ position and at the C₁₄ position, and ¹³C simultaneously in positions of C₁₄ and C₁₅. Each isotopic BR sample was examined not only in H₂O, but also in D₂O, which places a ²H at the Schiff base nitrogen of the retinal. In addition, PR/CARS data were recorded from each isotopic BR sample following either light adaptation [i.e. the BR sample contained a single retinal isomer (all-trans, 15-anti or BR-570)] or dark adaptation [i.e. the BR sample contained a mixture of comparable amounts of retinal isomers (BR-570 and 13-cis, 15-syn or BR-548)]. Excellent agreement was found between the vibrational features observed by PR/CARS and those obtained from spontaneous resonance Raman measurements from the same isotopically substituted BR pigments. Several new vibrational features were also found from the PR/CARS data. Vibrational Raman data from three of the isotopic BR samples in D₂O are reported for the first time.

The photocycle of bacteriorhodopsin (BR) regenerated with all-trans-9-demethylretinal was investigated by time-resolved rapid-scan Fourier transform infrared difference spectroscopy, by static lowtemperature difference spectroscopy at 80, 170, and 213 K and by static steady-state difference spectroscopy at 278 K. In addition, the formation and decay of M intermediate was monitored at 412 nm with conventional flash photolysis experiments. Our data show that the removal of the 9-methyl group strongly changes the photocycle of BR. The reaction cycle is slowed down about 250-fold. The photoreaction is characterized by a slow rise of the M intermediate and by a very long-lived N intermediate. No O intermediate could be observed. The low-temperature spectra indicate that already at 80 K a KL-like photoproduct is formed. L can be obtained as in native BR at 170 K, but its decay appears to be inhibited, since it can still be observed at 213 K and high pH, in addition to the M intermediate. As in native BR, the 15-hydrogen out-of-plane modes of the L and N intermediates (observed in ²H₂O) are very similar. Evidence for the existence of three N substates which differ in the protonation state of Asp96 and in the amide I bands is presented. This is explained by the extremely slowed-down reisomerization of the chromophore. The results are discussed with respect to alterations in the chromophore-protein interaction, caused by the removal of the 9-methyl group.

Deprotonation/protonation processes involving the retinal Schiff base and the Asp⁸⁵ residue play dominant roles in the light-induced proton pump of bacteriorhodopsin (bR). Although the pKa values of these two moieties in unphotolyzed bR are well established, the kinetics of the respective titrations in the native pigment are difficult to interpret, primarily due to the extreme (nonphysiological) pK_a values of the two moieties (12.2 ± 0.2 and 2.7, in 0.1 M NaCl, for the Schiff base and for Asp⁸⁵, respectively). These difficulties are circumvented by applying stopped-flow techniques, time resolving the titrations of several artificial bRs in which the p values of the above two residues are substantially modified: 13- CF₃ bR, pATa (Schiff base) = 8.2 ± 0.2; 13-demethyl-11,14-epoxy bR, pK_a (Schiff base) = 8.2 ± 0.1 (in 0.1 M NaCl); aromatic bR, p (Asp⁸⁵) = 5.2 ±0.1 (in water). The R82Q bR mutant, pK_a (Asp⁸⁵) = 7.2 was also employed. A major objective was to verify whether the basic relationships of homogeneous kinetics obeyed by elementary acid/base systems in solution (primarily, the possibility to express the equilibrium constant as the ratio of the forward and back rate constants) are also obeyed by the Schiff base and Asp⁸⁵ moieties. We found that this is the case for the Schiff base in the pH range between 7 and 9 but not at lower pH. These observations led to the conclusion that the Schiff base is titrable from the outside medium via a proton channel, which becomes saturated, and thus rate determining, below pH ss 7. The observed protonation rate constant in the pH = 7-9 range is K = 6.0 x 10⁷ M^-1 s^-1, implying a reactivity that is lower by 3 orders of magnitude as compared to the diffusion-controlled rate constant of an elementary acid/base in homogeneous solutions. In the case of Asp⁸⁵, fca could not be directly determined. The titration rates observed in the case of pigment IV are, however, consistent with a model in which the Schiff base and Asp⁸⁵ are exposed to the extracellular side via the same proton channel. It is suggested that the rate-determining step in proton translocation via this channel is a transfer between Asp⁸⁵ and the outside, rather than between Asp⁸⁵ and the Schiff base. This conclusion applies independently of whether Asp⁸⁵ is protonated or non-protonated. The results are relevant to basic questions related to the proton pump mechanism in bR, primarily (a) the exposure direction (to the outside or to the inside of the cell) of the Schiff base and of Asp⁸⁵ in unphotolyzed bR and (b) the nature of the still unidentified protein residue (XH) whose proton is translocated to the outside during the bacteriorhodopsin photocycle. We conclude that, in variance with the Schiff base in unphotolyzed bR or with Asp⁸⁵ (in photolyzed or unphotolyzed bR), during the photocycle the XH moiety is highly exposed to the outside medium. More generally, our study bears on the basic problem concerning the relationship between the kinetics of the titration of protein residues and their respective (\u201dthermodynamic\u201d) equilibrium constants.

Abstract aIsorhodopsin, an artificial visual pigment with a 9cis4,5dehydro5,6dihydro(a)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)like intermediate absorbing at ca 495 nm was detected below 55 K, a blueshifted intermediate (BSI)like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6dihydroisorhodopsin. As in native rhodopsin, Batho is the first intermediate detected in aisorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. aIsorhodopsin behaves similarly to 5,6dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C₅C₆ double bond. The transition temperature for BSI formation is higher in aisorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.

Nanosecond laser photolysis measurements were conducted on the cone-type visual pigment P521 in digitonin extracts of Tokay gecko (Gekko gekko) retina containing physiological chloride ion levels and also on samples which had been chloride depleted or which contained high levels (4 M) of chloride. Absorbance difference spectra were recorded at a sequence of time delays from 30 ns to 60 µs following excitation with a pulse of either 532- or 477-nm actinic light. Global analysis showed the kinetic decay data for gecko pigment P521 to be best fit by two exponential processes under all chloride conditions. The initial photoproduct detected had a broad spectrum characteristic of an equilibrated mixture of a Batho P521 intermediate with its blue-shifted intermediate (BSI P521) decay product. The first exponential process was assigned to the decay of this mixture to the Lumi P521 intermediate. The second exponential process was identified as the decay of Lumi P521 to Meta I P521. The initial photoproduct's spectrum exhibited a strong dependence on chloride concentration, indicating that chloride affects the composition of the equilibrated mixture of Batho P521 and BSI P521. These results suggest that the affinity for chloride is reduced ~5-fold in the Batho P521 intermediate and ~50-fold in the BSI P521 intermediate. Chloride concentration also affects the apparent decay rate of the equilibrated mixture. When the apparent decay rate is corrected for the composition of the equilibrated mixture, a relatively invariant microscopic rate constant is obtained for BSI decay (k = 1/55 ns^-1). The rate constant obtained agrees with the value observed for the synthetic 9-m-α-retinal gecko pigment which has a completely forward-shifted equilibrium mixture, indicating that the BSI decay is controlled by a relaxation process independent of chromophore and chloride concentration and thus presumably characteristic of the gecko opsin protein.

The picosecond dynamics of the photoreaction of an artificial bacteriorhodopsin (BR) pigment containing a retinal in which a five-membered ring spans the C-12 to C-14 positions of the polyene chain (BR5.12) is examined by using time-resolved absorption and fluorescence and resonance Raman spectroscopy. The ring within the retinal chromophore of BR5.12 blocks the C-13=C-14 isomerization proposed to be a primary step in the energy storage/transduction mechanism in the BR photocycle. Relative to the native BR pigment (BR-570), the absorption spectrum of BR5.12 is red-shifted by 8 nm. The fluorescence spectrum of BR5.12 closely resembles that of BR-570 although the relative fluorescence yield is higher (≃10-fold). Picosecond transient absorption (4-ps pulses, 568-662 nm) measurements reveal an intermediate absorbing to the red side of BR5.12. Kinetic fits show that the red-absorbing intermediate appears within

The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally-modified retinal chromophore with a six-membered ring beginning at C₉ to bridge the C₉=C₁₀-C₁₁ region of the polyene chain (BR6.9) are measured by picosecond transient absorption (PTA). Time-dependent absorption intensity and spectral changes in the 560-670 nm region are monitored for delays as long as 54 ns after the 4-ps, 573-nm excitation of BR6.9. Within 600 nm) of the BR6.9 spectrum. The first intermediate decays with a time constant of 5 ± 1 ps to form the second, but no other absorption changes are found during the remainder of the initial 54-ns period of the BR6.9 photoreaction. Since these PTA properties are generally analogous to those measured in the native BR photocycle for J-625 and K-590, the two BR6.9 intermediates are denoted J6.9 and K6.9, respectively. The low-power energy, resonance Raman (RR) spectrum of ground-state BR6.9 is significantly different from that of native BR-570, thereby confirming that these PTA data are assignable to BR6.9 and its photoreaction alone and not to native BR species (BR-570 could remain in the reconstituted sample as a contaminant). The C-C stretching band structure in the RR spectrum of BR6.9 is similar to that of another of the artificial BR pigments in which the six-membered ring is incorporated to bridge the C₁₁=C₁₂-C₁₃ bonds, namely BR6.11. Mechanistically, these results demonstrate that restricted motion in the C₉=C₁₀-C₁₁ region does not change that part of the BR6.9 photoreaction involved in forming J6.9 and K6.9 but does alter the rate at which the J to K transformation occurs. A molecular model correlating the primary events and their rates in the native BR photocycle with those appearing in the BR6.9 photoreaction, as well as with other artificial BR pigments containing carbon rings, is presented.

The nonlinear optical properties of the bacteriorhodopsin chromophore in the bR568 and K states are investigated by second harmonic generation. The comparison of amplitudes and phases of the second-order nonlinear optical polarizabilities of the retinal chromophore in the two states has revealed a noticeable increase of the induced dipole of the retinal as a result of the bR568 → K transition. The results have been explained in terms of recent theoretical understandings of the nonlinear optical properties of polyenes. Within the context of these understandings we have discussed the molecular origins of the light-induced color changes and the possible mechanism of photon energy storage observed in this protein.

The early stages of the bacteriorhodopsin photocycle, including the J₆₂₅, K₅₉₀, and L₅₅₀ intermediates and the role of water molecules within the protein interior, are studied by means of molecular dynamics simulations. Our calculations examine two models for the excited state potential surface governing the observed all-trans → 13-cis photoisomerization: one surface hindering a C₁₄-C₁₅ single-bond corotation and the other surface allowing such corotation. The investigations use as a starting structure a model of bacteriorhodopsin based on electron-microscopy studies and subsequent molecular dynamics refinement. The following questions are addressed: How does the binding site guide retinal's photoisomerization? How does the photoisomerization depend on features of the excited state potential surface? Can one recognize a J₆₂₅ intermediate? How does water participate in the early part of the pump cycle? How is the initial photoreaction affected by a lowering of temperature? To model the quantum yield, i.e., the dependence of the dynamics on initial conditions, 50 separate isomerization trials are completed for each potential surface, at both 300 and 77 K, the trials being distinguished by different initial, random velocity distributions. From these trials emerge, besides all-trans-retinal, three different photoproducts as candidates for the K₅₉₀ intermediate: (1) 13-cis-retinal, with the Schiff base proton oriented toward Asp-96; (2) 13-cis-retinal, highly twisted about the C₆-C₇ bond, with the Schiff base proton oriented perpendicular to the membrane normal; (3) 13,14-dicis-retinal, with the Schiff base proton oriented toward the extracellular side. Two candidates for the K₅₉₀ intermediate, case 2 and case 3 above, were subjected to simulated annealing to determine corresponding L₅₅₀ structures. We suggest that photoproduct 2 above most likely represents the true K₅₉₀ intermediate. Water molecules near the Schiff base binding site are found to play a crucial role in stabilizing the K₅₉₀ state and in establishing a pathway for proton transfer to Asp-85.

The M stage in the photocycle of bacteriorhodopsin (bR), a key step in its light-induced proton pump mechanism, is studied in water/glycerol suspensions over the temperature range between 20 and −60 °C. The biexponential decay of M is analyzed for wild-type (WT) bR and for its D96N, Y185F, and D115N mutants, at various pH values, according to the scheme: bR →(hv) L → M → (k₁, k_-1) N→ (k₂) bR. The analysis leads to the conclusion that the N state is generated, with analogous rate parameters, in all cases, including the D96N mutant. Another approach involves probing the M state, generated by steady-state illumination at −60 °C, by fast cooling to −180 °C. Subsequent irradiation with blue light, followed by gradual warming up, induces the M→ (hv) M→ bR→ bR sequence of reactions. On the basis of characteristic difference spectra and transition temperatures observed for the M→ bR process, it is concluded that the initially observed M state at −60 °C, denoted as {M}_a, is composed of three (or four) equilibrated substates, M_I, M_II, M_III, and M_IV. During the M→ N equilibration, which corresponds to the fast phase of the M decay, {M}_a transforms into a second state, (M}_b, in which M_III has been replaced by a fifth M substate, denoted as M_v. M_v is identified as the protein state in which an appropriate structural change allows reprotonation of the Schiff base, generating the N state. The low-temperature heterogeneity in M is discussed in terms of the two M states (M₁ and _M2) previously postulated [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241] for the room temperature photocycle. The following conclusions are derived for both low and room temperature photocycles: (a) The M population is highly heterogeneous and pH dependent, (b) At least three transitions are observed between the initially formed M state and the M state that is equilibrated with N. These are assigned to protein conformational changes and to water molecule rearrangements, (c) In an aqueous suspension of WT bR at room temperature, the Schiff base reprotonation is controlled by D96. However, our results show that the formation and stability of the N state do not require the D96 residue. Moreover, at low temperatures, the (M}_a→ {M}_b protein structural transformation, which has not yet been resolved at room temperature, becomes the rate-determining step in the protonation of the Schiff base.

The light-induced proton pumping activity of bacteriorhodopsin (bR) is based on the photocycle of its light-adapted all-trans-retinal protein pigment. The photocycle of the 13-cis pigment lacks the M intermediate (which carries a deprotonated retinal Schiff base, characteristic of the all-trans photocycle) and is not associated with proton release and uptake. Aiming at establishing the reasons for the lack of light-induced Schiff base deprotonation and proton pumping in 13-cis-bR, we carried out pulsed-laser and continuous excitation experiments with artificial 13-cis-bR pigments derived from 13-demethylretinal, 13-demethyl-14-fluoro-bR, and 13-demethyl-12,14-difluoro-bR. Pulsed-laser photolysis shows that both M formation and proton pumping are restored in 13-cis-13-demethyl-bR by raising the pH to 8.5-9. M formation, but not proton pumping, is restored at neutral pH by 14-fluorine substitution. Continuous-illumination experiments lead, in all cases, to the generation of extremely long-lived (minutes to hours) M photoproducts. We show that such species are due to secondary photoreactions of late intermediates of the primary photolysis. Feasible mechanisms accounting for Schiff base deprotonation in the all-trans photocycle, but not in that of 13-cis-bR, are considered. Our findings favor a mechanism which attibutes the lack of light-induced Schiff base deprotonation of 13-cis-bR to an insufficient change in the relative pK_a of the donor (Schiff base) and acceptor (probably Asp-85) groups and/or to a high activation barrier for the proton transfer. The required change in relative pK_as may be achieved either by deprotonation of a protein moiety (YH) with pK_a ≈ 8.5 or by fluorine substitution at position 14. Similarly, both YH titration and 14-fluorine substitution may reduce the barrier for proton transfer by affecting H-bonding interactions in the vicinity of the Schiff base linkage. The lack of proton release and uptake in the photocycle of 13-cis-13-demethyl-14-fluoro-bR, despite the presence of an M intermediate, is discussed. It appears that Schiff base deprotonation does not essentially imply proton release and uptake. Our conclusions bear on the molecular mechanism of the photocycle and of proton pumping in all-trans-bR.

Following light absorption, at neutral pH the bacteriorhodopsin mutant Y57N does not show Schiff base deprotonation (no M intermediate) or proton pumping activity. We reasoned that this might be due to improper ΔpK_a between the proton-donating Schiff base and the proton-accepting Asp-85 after light absorption. To test this, we reduced the intrinsic pK_a of the protonated Schiff base in the pigment (and thus in the photointermediates) by replacing the retinal chromophore with an analogue, 14-F retinal. This substitution restores light-induced M formation, strongly suggesting that light-induced Schiff base deprotonation is accomplished by lowering its pK_a during the photochemical cycle. Thus, while it is generally accepted that the Schiff base deprotonation during the photocycle takes place because of the light-induced reduction in its pK_a, we provide here the first experimental evidence of this phenomenon.

The structure of bacteriorhodopsin based on electron microscopy (EM) studies, as provided in Henderson et al. (1990), is refined using molecular dynamics simulations. The work is based on a previously refined and simulated structure which had added the interhelical loops to the EM model of bR. The present study applies an all-atom description to this structure and constraints to the original Henderson model, albeit with helix D shifted. Sixteen waters are then added to the protein, six in the retinal Schiff base region, four in the retinal-Asp-96 interstitial space, and six near the extracellular side. The root mean square deviation between the resulting structure and the Henderson et al. (1990) model measures only 1.8 Å. Further simulations of retinal analogues for substitutions at the 2- and 4-positions of retinal and an analogue without a β-ionone ring agree well with observed spectra. The resulting structure is characterized in view of bacteriorhodopsin's function; key features are (1) a retinal Schiff base-counterion complex which is formed by a hydrogen bridge network involving six water molecules, Asp-85, Asp-212, Tyr-185, Tyr-57, Arg-82, and Thr-89, and which exhibits Schiff base nitrogen-Asp-85 and -Asp-212 distances of 6 and 4.6 Å; (2) retinal assumes a corkscrew twist as one views retinal along its backbone; and (3) a deviation from the usual α-helical structure of the cytoplasmic side of helix G.

The factors that red shift the absorption maximum of the retinal Schiff base chromophore in the M₄₁₂ intermediate of bacteriorhodopsin photocycle relative to absorption in solution were investigated using a series of artificial pigments and studies of model compounds in solution. The artificial pigments derived from retinal analogs that perturb chromophoreprotein interactions in the vicinity of the ring moiety indicate that a considerable part of the red shift may originate from interactions in the vicinity of the Schiff base linkage. Studies with model compounds revealed that hydrogen bonding to the Schiff base moiety can significantly red shift the absorption maximum. Furthermore, it was demonstrated that although strans ringchain planarity prevails in the M₄₁₂ intermediate it does not contribute significantly (only ca 750 cm⁻¹) to the opsin shift observed in M₄₁₂. It is suggested that in M₄₁₂, the Schiff base linkage is hydrogen bonded to bound water and/or protein residues inducing a considerable red shift in the absorption maximum of the retinal chromophore.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.

The picosecond molecular dynamics in an artificial bacteriorhodopsin (BR) pigment containing a structurally modified all-trans retinal chromphore with a six-membered ring bridging the C11=C12-C13 positions (BR6.11) are measured by picosecond transient absorption and picosecond time-resolved fluorescence spectroscopy. Time-dependent intensity and spectral changes in absorption in the 570650-nm region are monitored for delays as long as 5 ns after the 7-ps, 573-nm excitation of BR6.11. Two intermediates, J6.11 and K6.11/1, both with enhanced absorption to the red (> 600 nm) of the BR6.11 spectrum are observed within approximately 50 ps. The J6.11 intermediate decays with a time constant of 12 +/- 3 ps to form K6.11/1. The K6.11/1 intermediate decays with an approximately 100-ps time constant to form a third intermediate, K6.11/2, which is observed through diminished 650-nm absorption (relative to that of K6.11/1). No other transient absorption changes are found during the remainder of the initial 5-ns period of the BR6.11 photoreaction. Fluorescence in the 650900-nm region is observed from BR6.11, K6.11/1, and K6.11/2, but no emission assignable to J6.11 is found. The BR6.11 fluroescence spectrum has a approximately 725-nm maximum which is blue-shifted by approximately 15 nm relative to that of native BR-570 and is 4.2 +/- 1.5 times larger in intensity (same sample optical density). No differences in the profile of the fluorescence spectra of BR6.11 and the intermediates K6.11/1 and K6.11/2 are observed. Following ground-state depletion of the BR6.11 population, the time-resolved fluroescence intensity monitored at 725 nm increases with two time constants, 12 +/- 3 and approximately 100 ps, both of which correlate well with changes in the picosecond transient absorption data.(ABSTRACT TRUNCATED AT 250 WORDS)

The back photoreaction of the M intermediate in the photocycle of bacteriorhodopsin is investigated both for the native pigment and its D96N mutant. The experimental setup is based on creating the M intermediate by a first pulse, followed by a (blue) laser pulse which drives the back photoreaction of M. Experiments are carried out varying the delay between the two pulses, as well as the temperature over the -25-degrees-C-20-degrees-C range. It is found that the kinetic patterns of the M back photoreaction change with time after the generation of this intermediate. The data provide independent evidence for the suggestion of a photocycle mechanism based on two distinct M intermediates. They are thus in keeping with the consecutive model of Varo and Lanyi (Biochemistry 30, 5016-5022; 1991), although they cannot exclude other models such as those based on branched or parallel cycles. More generally, we offer a ''photochemical'' approach to discriminating between intermediate stages in the photocycle which does not depend on spectroscopic and/or kinetic data. While markedly affecting the rate of the M --> N transition in the photocycle, the rate of the thermal step in the back photoreaction of M, at both room and low temperatures, is not significantly affected by the D96N mutation. It is proposed that while Asp 96 is the Schiff-base protonating moiety in the M --> N transition, another residue (most probably Asp 85) reprotonates the Schiff base following light absorption by M.

Bacteriorhodopsin (bR) has been biosynthetically prepared with lysine deuterated at its o carbon (Cα - H). The labeled membranes containing bR were investigated by difference Fourier transform infrared (FTIR) spectroscopy. It has been derived from K/bR and M/bR difference spectra (K and M are photocycle intermediates) that several bands previously assigned to the retinal chromophore are coupled to the Cα - H. The vibrational modes that exhibit this coupling are principally associated with C15 - H and N - H vibrations. [Cα-²H]Lysine-labeled bR was fragmented enzymatically, and bR structures were regenerated with the Cα - ²H label either on lysine-216 and -172 or on the remaining five lysine residues of the protein. FTIR studies of the regenerated bR system, together with methylation of all lysines except the active-site lysine, reveal that the changes observed due to backbone labeling arise from the active-site lysine. The intensity of the C15 - H out-of-plane wag is interpreted as a possible indication of a twist around the C15=N bond.

Abstract The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both darkadapted (DA) and lightadapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13cis) and Mtrans, are observed. However, the artificial DA pigments have a lower 13d.s content than native DA bR (˜ 66%) while the corresponding LA pigments have a much higher 13cis content (1169%) than native LA bR (

FTIR studies of the BSI photoproduct and of the later intermediates lumirhodopsin and metarhodopsin-I of 5,6-dihydroisorhodopsin are reported on. Evidence is presented that in the BSI intermediate the retinal chromophore adopts a relaxed conformation in contrast to the bathorhodopsin intermediate of native rhodopsin. Whereas for the modified pigment, the Schiff base C=N stretching mode and its deuteration-induced isotopic shifts are similar to those of unmodified isorhodopsin, the corresponding values of the photoproducts differ. In addition, alterations in the carbonyl spectral range are observed (protonated carboxyl groups and amide-I). This shows that the chromophore-protein interaction is influenced by this modification. Some molecular events of the thermal decay of the bleached pigment occur at lower temperatures or even at an earlier intermediate of the photoreaction than in native rhodopsin.

Protonated Schiff bases of retinal (RSBH⁺), of its (planar) linear polyene analogue 1,1-didemethylretinal (LRSBH+), and of an analogous cyanine dye (Cy^III) are submitted to pulsed laser photolysis over a range of solvents and temperatures. Transient phenomena observed with the Cy^IIIdye are attributed to trans → cis isomerization, followed by secondary excitation which induces rotation about an additional bond. In the cases of RSBH⁺ and LRSBH⁺ (photostationary mixtures of cis- trans isomers), laser excitation of deaerated solutions leads to the observation of triplet states. The latter are formed via intersystem crossing (ISC) from a short-lived excited state, generated by multiple excitations of the ground state during the (same) intense laser pulse. 0₂ saturation of the solutions suppresses the ISC route, giving rise to a short-lived phototransient observed at low temperatures, which is identified as a C-C conformer. The observations are discussed in light of the possibility that C-C conformers may play a role in the photocycles of visual rhodopsins and of bacteriorhodopsin. Experiments were also per formed with an artificial bacteriorhodopsin pigment (bRcy) carrying a cyanine chromophore analogous to Cy^III. A single photointermediate (bR_cy/I), reminiscent of the K phototransient of bR, is observed and is attributed to a cis isomer. The low activation and preexponential parameters which characterize the thermal relaxation of bR_cy/I are discussed in terms of cis trans isomerization in a rhodopsin binding site. The results bear on the thermal 13-cis all-trans relaxation in the final stages of the bR photocycle and on the inefficiency of a back (all-trans → 11-cis) reaction at the early (bathorhodopsin) stage of the visual photocycle.

The absorption maximum (568 nm) of light-adapted bacteriorhodopsin bR568 undergoes reversible changes after acidification. At pH 2.9, the absorption shifts to 605 nm (forming bR605) and it blue shifts to 565 nm, after further acidification to pH approximately 0.5 (forming bR565). Molecular models accounting for such acid-induced changes are relevant to the structure and function of bacteriorhodopsin. In the present study we approached the problem by applying artificial bR pigments based on selectively modified synthetic retinals. This may allow direct identification of the specific regions in the retinal binding site where the above changes in the protein-retinal interactions take place. We investigated the spectroscopic effects of acid in a variety of artificial pigments, including cyaninelike retinals, retinals bearing bulky groups at C4, short polyenes, and retinals in which the beta-ionone ring was substituted by aromatic rings. The results provide direct evidence for the hypothesis that the generation of bR605 is due to changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. The second transition (to bR565) was not observed in artificial pigments bearing major changes in the ring structure of the retinal. Two approaches accounting for this observation are presented. One argues that the generation of bR565 is associated with acid-induced changes in retinal-protein interactions in the vicinity of the retinal ring. The second involves changes in polyene-opsin interactions in the vicinity of the Schiff base linkage.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors affecting the C=N stretching frequency of protonated retinal Schiff base (RSBH⁺) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of RSBH⁺ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the 0=N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C=N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C=N stretching and C=NH bending vibration. The C=N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C=N stretching frequency of 1640 cm^-1 and the deuterium isotope effect of 17 cm^-1 observed in this pigment. Conversion of bR to the photochemically induced intermediate K₆₁₀ involves environmental perturbation in the vicinity of the C=N linkage, lowering the C=N stretch energy. The C=N stretching frequency (1660 cm^-1) observed for rhodopsin indicates very effective hydrogen bonding with the Schiff base counteranion and/or effective solvation by protein dipoles or residual water.

Factors influencing the Pka value of retinal orotonated Schiff base (RSBH⁺l are examined by using fluorinated alcohols and series of retinals bearing nonconjugated positive charges along the polyene. It is shown that the effective pK_a of RSBH⁺ is increased by a solvent, forming a strong hydrogen bond, that stabilizes the anion but weakly interacts with the Schiff-base proton. A positive charge in the vicinity of the Schiff-base linkage markedly reduces the effective pK_a. The effect is significantly enhanced in fluorinated alcohols in which positive charges are weakly solvated. It is suggested that drastic pK_a reduction might take place during bacteriorhodopsin (bR) photocycle either by elimination of hydrogen-bonding stabilization or by a positive charge approaching the Schiff-base linkage. Weak solvation of the positively charged Schiff-base nitrogen (relative to ethanol solution) and strong solvation with its counterion lead to a red shift in the absorption maximum of retinal protonated Schiff base up to ca. 2400 cm^-1 in hexafluoro-2-propanol relative to ethanol. This mechanism of introducing red shift in the absorption maximum of RSBH⁺ might play a role in determining part of the opsin shift found in bR and the red shift observed in the transformation from the bR₅₇₀ to K610 intermediate following light absorption. Nonconjugated positive charges shift the absorption maximum of RSBH⁺. Their influence is further enhanced with fluorinated alcohols as solvents.

Artificial bacteriorhodopsin (bR) pigments based on synthetic retinal analogues with selectively blocked single and double bonds were prepared. It was shown that rotations around single bonds C₁₂-C₁₃ and C₁₀-C₁₁ and isomerizations of C₁₁=C₁₂ and C₉=C₁₀ are not required either for initiating the photocycle of all-trans-bR or for forming its M₄₁₂ intermediate. The results are discussed in light of mechanisms for the primary event (based on the C₁₃=C₁₄ isomerization) involving a concerted double-bond and single-bond rotation around adjacent C,C bonds. Similarly, the photoreaction of the 13-cis isomer of bacteriorhodopsin does not require isomerization about the C₁₁=C₁₂ double bond or rotation around C₁₂-C₁₃. It is also shown that 13-cis ⇔ all-trans (light-dark adaptation) reaction of bacteriorhodopsin does not involve additional rotations or isomerizations involving the C₉-C₁₃ section of the molecule.

For the first time linear dichroism spectroscopy has been extended to the picosecond time regime. 11 -cis retinal, all-trans retinal and l,8-diphenyl-l,3,5,7-octatetraene (DPOT) are incorporated into polyethylene films and oriented by stretching the films. By measuring picosecond transient absorption spectra polarized parallel and perpendicular to the stretching direction an calculating the dichroic ratio we get informations about the molecular geometry in excited singlet and triplet states. The results may have relevance to vision. For the first time linear dichroism spectroscopy has been extended to the picosecond time regime. 11 -cis retinal, all-trans retinal and l,8-diphenyl-l,3,5,7-octatetraene (DPOT) are incorporated into polyethylene films and oriented by stretching the films. By measuring picosecond transient absorption spectra polarized parallel and perpendicular to the stretching direction and calculating the dichroic ratio we get informations about the molecular geometry in excited singlet and triplet states. The results may have relevance to vision vision.

The influence of external positive charges on the absorption maximum of protonated retinal Schiff base is markedly enhanced by weakening the interaction between the external positive charge and its counterion.

Do the changes in ν(CC) in visual pigments and bacteriorhodopsin orginate from interaction of the chromophore with charges in the protein moiety? This question was investigated by FTIR investigations on the model compounds 13. That ν(CC) in them is significantly higher than in the unprotonated parent compounds suggests that the answer to the question is \u201cyes\u201d. (Figure Presented.)

A number of retinoid derivatives have been synthesized for use as labels for cellular retinol-binding protein. Introduction of substituents abolished the binding of the derivatives to the protein, except in the case of the photo-reactive derivative, 4-azidoretinol. This compound was found to compete successfully with all-trans-retinol for binding to cellular retinol-binding protein, with a high relative binding affinity. Irradiation of a complex of 4-azidoretinol and a semi-purified preparation of cellular retinol-binding protein from liver resulted in a firm attachment stable to SDS-gel electrophoresis. It is therefore suggested that the irradiated product is held together covalently. A method for the synthesis of 4-azidoretinol is described.

A blue shift was observed in the absorption maxima of protonated retinal Schiff base. The shift is due to electrostatic interaction of a non-conjugated positive charge, located in the vicinity of the ionone moiety, with the polyene chromophore.

Large shifts of the maxima in the absorption spectra of protonated retinal Schiff bases and their benzenoid analogues were observed on changing the counter ion from CF₃CO₂^- to Cl^-.

The title compounds were synthesized and their dynamic properties investigated by variable temperature 1H NMR. Each of these compounds gave separate signals, at low temperature, for ring A conformers. The temperature-dependent spectra allowed the determination of the activation parameters characteristic of ring A chair-chair interconversion. The free energies of activation (Δ G∗@r@n) for the chair inversion in 4,4-dimethylvitamin D3 (4a), 4,4-dimethyl-1α-hydroxyvitamin D3 (5), and 4,4-dimethyl-lα-hydroxyepivitamin D3 (6) were found to be 10.1, 11.0, and 12.0 kcal/mol, respectively. The 13C NMR spectrum of 4a was recorded at room temperature and at low temperature (ca. -90 °C). The chemical shift separation of the two observed C3 signals, at low temperature, was used for conformational analysis.

Complete and self-consistent assignment of the ¹³C NMR spectra of the C₃-epimeric pairs of vitamin D₃, trans-vitamin D₃, its keto analogue, and dihydrotachysterol₂ were made. The conformational flexibility of ring A in these compounds and their esters was investigated using the ¹³C as well as₁H NMR data. The merits and the complementary nature of each approach are discussed in terms of the accuracy and reliability of the results. The relative excess of the equatorial conformer in the C₃ epimeric pairs of vitamin D₃ and its analogues was determined by both methods. This analysis indicates that in the case of vitamin D₃ and its C₃ epimer the methylene group affects the equilibrium population by stabilizing the chair conformation where the hydroxy group is axially oriented.