Publications

The remarkable ability of natural proteins to conduct electricity in the dry state over long distances remains largely inexplicable despite intensive research. In some cases, a (weakly) exponential length-attenuation, as in off-resonant tunneling transport, extends to thicknesses even beyond 10 nm. This report deals with such charge transport characteristics observed in self-assembled multilayers of the protein bacteriorhodopsin (bR). ≈7.5 to 15.5 nm thick bR layers are prepared on conductive titanium nitride (TiN) substrates using aminohexylphosphonic acid and poly-diallyl-dimethylammonium electrostatic linkers. Using conical eutectic gallium-indium top contacts, an intriguing, mono-exponential conductance attenuation as a function of the bR layer thickness with a small attenuation coefficient β ≈ 0.8 nm⁻¹ is measured at zero bias. Variable-temperature measurements using evaporated Ti/Au top contacts yield effective energy barriers of ≈100 meV from fitting the data to tunneling, hopping, and carrier cascade transport models. The observed temperature-dependence is assigned to the protein-electrode interfaces. The transport length and temperature dependence of the current densities are consistent with tunneling through the proteinprotein, and protein-electrode interfaces, respectively. Importantly, the results call for new theoretical approaches to find the microscopic mechanism behind the remarkably efficient, long-range electron transport within bR.

Animal vision depends on opsins, a category of G protein-coupled receptor (GPCR) that achieves light sensitivity by covalent attachment to retinal. Typically binding as an inverse agonist, 11-cis retinal photoisomerizes to the all-Trans isomer and activates the receptor, initiating downstream signaling cascades. Retinal bound to bistable opsins isomerizes back to the 11-cis state after absorption of a second photon, inactivating the receptor. Bistable opsins are essential for invertebrate vision and nonvisual light perception across the animal kingdom. While crystal structures are available for bistable opsins in the inactive state, it has proven difficult to form homogeneous populations of activated bistable opsins either via illumination or reconstitution with all-Trans retinal. Here, we show that a nonnatural retinal analog, all-Trans retinal 6.11 (ATR6.11), can be reconstituted with the invertebrate bistable opsin, Jumping Spider Rhodopsin-1 (JSR1). Biochemical activity assays demonstrate that ATR6.11 functions as a JSR1 agonist. ATR6.11 binding also enables complex formation between JSR1 and signaling partners. Our findings demonstrate the utility of retinal analogs for biophysical characterization of bistable opsins, which will deepen our understanding of light perception in animals.

Discovered over 50 years ago, bacteriorhodopsin is the first recognized and most widely studied microbial retinal protein. Serving as a light-activated proton pump, it represents the archetypal ion-pumping system. Here we compare the photochemical dynamics of bacteriorhodopsin light and dark-adapted forms with that of the first metastable photocycle intermediate known as \u201cK\u201d. We observe that following thermal double isomerization of retinal in the dark from bio-active all-trans 15-anti to 13-cis, 15-syn, photochemistry proceeds even faster than the ~0.5 ps decay of the former, exhibiting ballistic wave packet curve crossing to the ground state. In contrast, photoexcitation of K containing a 13-cis, 15-anti chromophore leads to markedly multi-exponential excited state decay including much slower stages. QM/MM calculations, aimed to interpret these results, highlight the crucial role of protonation, showing that the classic quadrupole counterion model poorly reproduces spectral data and dynamics. Single protonation of ASP212 rectifies discrepancies and predicts triple ground state structural heterogeneity aligning with experimental observations. These findings prompt a reevaluation of counter ion protonation in bacteriorhodopsin and contribute to the broader understanding of its photochemical dynamics.

The function of microbial as well as mammalian retinal proteins (aka rhodopsins) is associated with a photocycle initiated by light excitation of the retinal chromophore of the protein, covalently bound through a protonated Schiff base linkage. Although electrostatics controls chemical reactions of many organic molecules, attempt to understand its role in controlling excited state reactivity of rhodopsins and, thereby, their photocycle is scarce. Here, we investigate the effect of highly conserved tryptophan residues, between which the all-trans retinal chromophore of the protein is sandwiched in microbial rhodopsins, on the charge distribution along the retinal excited state, quantum yield and nature of the light-induced photocycle and absorption properties of Gloeobacter rhodopsin (GR). Replacement of these tryptophan residues by non-aromatic leucine (W222L and W122L) or phenylalanine (W222F) does not significantly affect the absorption maximum of the protein, while all the mutants showed higher sensitivity to photobleaching, compared to wild-type GR. Flash photolysis studies revealed lower quantum yield of trans-cis photoisomerization in W222L as well as W222F mutants relative to wild-type. The photocycle kinetics are also controlled by these tryptophan residues, resulting in altered accumulation and lifetime of the intermediates in the W222L and W222F mutants. We propose that protein-retinal interactions facilitated by conserved tryptophan residues are crucial for achieving high quantum yield of the light-induced retinal isomerization, and affect the thermal retinal re-isomerization to the resting state.

The finding that electronic conductance across ultrathin protein films between metallic electrodes remains nearly constant from room temperature to just a few degrees Kelvin has posed a challenge. We show that a model based on a generalized Landauer formula explains the nearly constant conductance and predicts an Arrhenius-like dependence for low temperatures. A critical aspect of the model is that the relevant activation energy for conductance is either the difference between the HOMO and HOMO1 or the LUMO+1 and LUMO energies instead of the HOMOLUMO gap of the proteins. Analysis of experimental data confirms the Arrhenius-like law and allows us to extract the activation energies. We then calculate the energy differences with advanced DFT methods for proteins used in the experiments. Our main result is that the experimental and theoretical activation energies for these three different proteins and three differently prepared solid-state junctions match nearly perfectly, implying the mechanisms validity.

Energy transfer from light-harvesting ketocarotenoids to the light-driven proton pump xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium¹ and a terrestrial cyanobacterium². Attempts to find carotenoids that bind and transfer energy to abundant rhodopsin proton pumps³ from marine photoheterotrophs have thus far failed⁴⁶. Here we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins using functional metagenomics combined with chromophore extraction from the environment. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet- or blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas may have a substantial effect on rhodopsin phototrophy in the worlds lakes, seas and oceans. However, the functional implications of our findings are yet to be discovered.

A way of modulating the solid-state electron transport (ETp) properties of oligopeptide junctions is presented by charges and internal hydrogen bonding, which affect this process markedly. The ETp properties of a series of tyrosine (Tyr)-containing hexa-alanine peptides, self-assembled in monolayers and sandwiched between gold electrodes, are investigated in response to their protonation state. Inserting a Tyr residue into these peptides enhances the ETp carried
their junctions. Deprotonation of the Tyr-containing peptides causes a further increase of ETp efficiency that depends on this residue's position. Combined results of molecular dynamics simulations and spectroscopic experiments suggest that the increased conductance upon deprotonation is mainly a result of enhanced coupling between the charged C-terminus carboxylate group and the adjacent Au electrode. Moreover, intra-peptide hydrogen bonding of the Tyr hydroxyl to the C-terminus carboxylate reduces this coupling. Hence, the extent of such a conductance change depends on the Tyr-carboxylate distance in the peptide's sequence.

The decades-long ultrafast examination of nearly a dozen microbial retinal proteins, ion pumps, and sensory photoreceptors has not identified structure-function indicators which predict photoisomerization dynamics, whether it will be sub-picosecond and ballistic or drawn out with complex curve-crossing kinetics. Herein, we report the emergence of such an indicator. Using pH control over retinal isomer ratios, photoinduced transient absorption is recorded in an inward proton pumping Antarctic microbial rhodopsin (AntR) for 13-cis and all-trans retinal resting states. The all-trans fluorescent state decays with 1 ps exponential kinetics. In contrast, in 13-cis it decays within ∼300 fs accompanied by continuous spectral evolution, indicating ballistic internal conversion. The coherent wave packet nature of 13-cis isomerization in AntR matches published results for bacteriorhodopsin (BR) and Anabaena sensory rhodopsin (ASR), which also accommodate both all-trans and 13-cis retinal resting states, marking the emergence of a first structure-photodynamics indicator which holds for all three tested pigments.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center. Bestrhodopsins are metastable and undergo photoconversion between red- and green-absorbing or green- and UVA-absorbing forms in the different variants. The retinal chromophore, in a unique binding pocket, photoisomerizes from all-trans to 11-cis form. Heterologously expressed bestrhodopsin behaves as a light-modulated anion channel.

Nanofluidics is an emerging hot field that explores the unusual behaviors of ions/molecules transporting through nanoscale channels, which possesses a broad application prospect. However, in situ probing bioactivity of functional proteins on a single-molecule level by a nanofluidic device has not been reported, and it is still a big challenge in the field. Herein, we reported a biological nanofluidic device with a single-protein sensitivity, based on natural proton-pumping protein, bacteriorhodopsin (bR), and a single SiNx nanopore. Nanofluidic single-molecule probing of bR proton-pumping activity and its light response were achieved under applied voltage of 0 V, by biologically self-powered steady-state ionic current nanopore sensing. Green-light irradiation of the device led to the monitoring of a steady-state proton current of ∼3.51 pA/per bR trimer, corresponding to charge density of 815 μC/cm² generated by each bR monomer, which far exceeded the previously reported value of 1.4 μC/cm². This finding and method would promote the development of artificial biological and hybrid nanofluidic devices in biosensing and energy conversion applications.

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?

Rhodopsin and carotenoids are two molecules that certain bacteria use to absorb and utilize light. Type I rhodopsin, the simplest active proton transporter, converts light energy into an electrochemical potential. Light produces a proton gradient, which is known as the proton motive force across the cell membrane. Some carotenoids are involved in light absorbance and transfer of absorbed energy to chlorophyll during photosynthesis. A previous study in Salinibacter ruber has shown that carotenoids act as antennae to harvest light and transfer energy to retinal in xanthorhodopsin (XR). Here, we describe the role of canthaxanthin (CAN), a carotenoid, as an antenna for Gloeobacter rhodopsin (GR). The non-covalent complex formed by the interaction between CAN and GR doubled the proton pumping speed and improved the pumping capacity by 1.5-fold. The complex also tripled the proton pumping speed and improved the pumping capacity by 5-fold in the presence of strong and weak light, respectively. Interestingly, when canthaxanthin was bound to Gloeobacter rhodopsin, it showed a 126-fold increase in heat resistance, and it survived better under drought conditions than Gloeobacter rhodopsin. The results suggest direct complementation of Gloeobacter rhodopsin with a carotenoid for primitive solar energy harvesting in cyanobacteria.

The research described in this report seeks to present proof-of-concept for a novel and robust platform for purification of antibody fragments and to define and optimize the controlling parameters. Purification of antigen-binding F(ab)2 fragments is achieved in the absence of chromatographic media or specific ligands, rather by using clusters of non-ionic detergent (e.g. Tween-60, Brij-O20) micelles chelated via Fe2+ ions and the hydrophobic chelator, bathophenanthroline (batho). These aggregates, quantitatively capture the F(ab)2 fragment in the absence or presence of E. coli lysate and allow extraction of only the F(ab)2 domain at pH 3.8 without concomitant aggregate dissolution or coextraction of bacterial impurities. Process yields range from 70 to 87% by densitometry. Recovered F(ab)2 fragments are monomeric (by dynamic light scattering), preserve their secondary structure (by circular dichroism) and are as pure as those obtained via Protein A chromatography (from a mixture of F(ab)2 and Fc fragments). The effect of process parameters on Ab binding and Ab extraction (e.g. temperature, pH, ionic strength, incubation time, composition of extraction buffer) are reported, using a monoclonal antibody (mAb) and polyclonal human IgGs as test samples.

We have recently described a non-chromatographic, ligand-free approach for antibody (Ab) purification based on specially designed [Tween-20:bathophenanthroline:Fe2+] aggregates. To assess the potential generality of this approach, a variety of detergents belonging to four nonionic detergent families (Tween, Brij, Triton and Pluronic) have now been studied. All surfactant aggregates led to high purity of the recovered Ab's (>95 %, by gel densitometry). Good overall Ab recovery yields were observed with Tween-20 (80-83 %), Brij-O20 (85-87 %) and Triton X-100 (87-90 %), while Pluronic F-127 was less efficient (42-53 %). Of additional importance is the finding that the process was performed by filtration rather than centrifugation, thereby allowing a continuous purification mode that led to the recovery of monomeric IgG, as determined by dynamic light scattering and preservation of Ab specificity as measured by ELISA. The amphiphilic chelator, bathophenanthroline (batho) was recycled almost quantitatively (95 %) by crystallization. Good IgG recovery yields of similar to 80 % were also observed when Ab concentrations were increased from 1 mg/mL to 3-5 mg/mL. Potential advantages of the purification platform for industrial downstream processing of therapeutic monoclonal antibodies, are discussed.

Multi-heme cytochromes (MHCs) are fascinating proteins used by bacterial organisms to shuttle electrons within, between, and out of their cells. When placed in solid-state electronic junctions, MHCs support temperature-independent currents over several nanometers that are 3 orders of magnitude higher compared to other redox proteins of similar size. To gain molecular-level insight into their astonishingly high conductivities, we combine experimental photoemission spectroscopy with DFT+ς current-voltage calculations on a representative Gold-MHC-Gold junction. We find that conduction across the dry, 3 nm long protein occurs via off-resonant coherent tunneling, mediated by a large number of protein valence-band orbitals that are strongly delocalized over heme and protein residues. This picture is profoundly different from the electron hopping mechanism induced electrochemically or photochemically under aqueous conditions. Our results imply that the current output in solid-state junctions can be even further increased in resonance, for example, by applying a gate voltage, thus allowing a quantum jump for next-generation bionanoelectronic devices.

We report the first observation of an efficient, native membrane conjugation mechanism via positively charged, linear oligo-amines. Clustering of membrane fragments relies on electrostatic interactions between the net negative charge of the membranes and the positively charged, water-soluble mediators. This conjugation principle is demonstrated with two different bacterial membranes in which are embedded either the intrinsic membrane protein (MP) bacteriorhodopsin (bR) or the more recently identified xanthorhodopsin (XR). As determined by their characteristic UV-vis absorption spectra and by circular dichroism, the MPs are not significantly perturbed by the oligo-amines carrying from +3 to +6 positive charges. Light microscopy and scanning electron microscope (SEM) imaging provide direct evidence for membrane conjugation. Process efficiency was found to be correlated with the net charge of the oligo-amine used. Membrane conjugation is accomplished within a wide range of pH values (7-2.5); is reversed by NaCl; and does not require the presence of a precipitant (e.g. PEG) nor Ca2+ ions. Some evidence for bilayer fusion is also observed, but only in the presence of the +6 oligo-amine analog.

A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1-2 (KS1-2), using the amphiphilic 4,4 ' -dinonyl-2,2 ' -dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal-chelator] complexes leads to purple, hexagonal crystals (50-75 mu m in size) of bR or pink, rectangular/square crystals (5-15 mu m) of KS1-2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.

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.

The primary sequence and secondary structure of a peptide are crucial to charge migration, not only in solution (electron transfer, ET), but also in the solid-state (electron transport, ETp). Hence, understanding the charge migration mechanisms is fundamental to the development of biomolecular devices and sensors. We report studies on four Aib-containing helical peptide analogues: two acyclic linear peptides with one and two electron-rich alkene-based side chains, respectively, and two peptides that are further rigidified into a macrocycle by a side bridge constraint, containing one or no alkene. ETp was investigated across Au/peptide/Au junctions, between 80 and 340 K in combination with the molecular dynamic (MD) simulations. The results reveal that the helical structure of the peptide and electron-rich side chain both facilitate the ETp. As temperature increases, the loss of helical structure, change of monolayer tilt angle, and increase of thermally activated fluctuations affect the conductance of peptides. Specifically, room temperature conductance across the peptide monolayers correlates well with previously observed ET rate constants, where an interplay between backbone rigidity and electron-rich side chains was revealed. Our findings provide new means to manipulate electronic transport across solid-state peptide junctions.

Understanding the factors affecting the stability and function of proteins at the molecular level is of fundamental importance. In spite of their use in bioelectronics and optogenetics, factors influencing thermal stability of microbial rhodopsins, a class of photoreceptor protein ubiquitous in nature are not yet well-understood. Here we report on the molecular mechanism for thermal denaturation of microbial retinal proteins, including, a highly thermostable protein, thermophilic rhodopsin (TR). External stimuli-dependent thermal denaturation of TR, the proton pumping rhodopsin of Thermus thermophilus bacterium, and other microbial rhodopsins are spectroscopically studied to decipher the common factors guiding their thermal stability. The thermal denaturation process of the studied proteins is light-catalyzed and the apo-protein is thermally less stable than the corresponding retinal-covalently bound opsin. In addition, changes in structure of the retinal chromophore affect the thermal stability of TR. Our results indicate that the hydrolysis of the retinal protonated Schiff base (PSB) is the rate-determining step for denaturation of the TR as well as other retinal proteins. Unusually high thermal stability of TR multilayers, in which PSB hydrolysis is restricted due to lack of bulk water, strongly supports this proposal. Our results also show that the protonation state of the PSB counter-ion does not affect the thermal stability of the studied proteins. Thermal photo-bleaching of an artificial TR pigment derived from non-isomerizable trans-locked retinal suggests, rather counterintuitively, that the photoinduced retinal trans-cis isomerization is not a pre-requisite for light catalyzed thermal denaturation of TR. Protein conformation alteration triggered by light-induced retinal excited state formation is likely to facilitate the PSB hydrolysis.

The chlorophyll-derivative chlorin e6 (Ce6) identified in the retinas of deep-sea ocean fish is proposed to play a functional role in red bioluminescence detection. Fluorescence and H-1 NMR spectroscopy studies with the bovine dim-light photoreceptor, rhodopsin, indicate that Ce6 weakly binds to it with mu m affinity. Absorbance spectra prove that red light sensitivity enhancement is not brought about by a shift in the absorbance maximum of rhodopsin. F-19 NMR experiments with samples where F-19 labels are either placed at the cytoplasmic binding site or incorporated as fluorinated retinal indicate that the cytoplasmic domain is highly perturbed by binding, while little to no changes are detected near the retinal. Binding of Ce6 also inhibits G-protein activation. Chemical shift changes in H-1-N-15 NMR spectroscopy of N-15-Trp labeled bovine rhodopsin reveal that Ce6 binding perturbs the entire structure. These results provide experimental evidence that Ce6 is an allosteric modulator of rhodopsin.

We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated "Engineered-micelles" built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe(2+)ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70-80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.

The incorporation of proteins as functional components in electronic junctions has received much interest recently due to their diverse bio-chemical and physical properties. However, information regarding the energies of the frontier orbitals involved in their electron transport (ETp) has remained elusive. Here we employ a new method to quantitatively determine the energy position of the molecular orbital, nearest to the Fermi level (E-F) of the electrode, in the electron transfer protein Azurin. The importance of the Cu(ii) redox center of Azurin is demonstrated by measuring gate-controlled conductance switching which is absent if Azurin's copper ions are removed. Comparing different electrode materials, a higher conductance and a lower gate-induced current onset is observed for the material with smaller work function, indicating that ETp via Azurin is LUMO-mediated. We use the difference in work function to calibrate the difference in gate-induced current onset for the two electrode materials, to a specific energy level shift and find that ETp via Azurin is near resonance. Our results provide a basis for mapping and studying the role of energy level positions in (bio)molecular junctions.

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.

Multi-heme cytochrome c (Cytc) proteins are key for transferring electrons out of cells, to enable intracellular oxidation to proceed in the absence of O-2. In these proteins most of the hemes are arranged in a linear array suggesting a facile path for electronic conduction. To test this, we studied solvent-free electron transport across two multi-heme Cytc-type proteins: MtrF (deca-heme Cytc) and STC (tetra-heme Cytc). Transport is measured across monolayers of these proteins in a solid state configuration between Au electrodes. Both proteins showed 1000x higher conductance than single heme, or heme-free proteins, but similar conductance to monolayers of conjugated organics. Conductance is found to be temperature-independent (320-80 K), suggesting tunneling as the transport mechanism. This mechanism is consistent with I-V curves modelling, results of which could be interpreted by having protein-electrode coupling as rate limiting, rather than transport within the proteins.

By comparing two-dimensional electronic spectroscopy (2DES) and Pump-Probe (PP) measurements on xanthorhodopsin (XR) and reduced-xanthorhodopsin (RXR) complexes, the ultrafast carotenoid-to-retinal energy transfer pathway is revealed, at very early times, by an excess of signal amplitude at the associated cross-peak and by the carotenoid bleaching reduction due to its ground state recovery. The combination of the measured 2DES and PP spectroscopic data with theoretical modelling allows a clear identification of the main experimental signals and a comprehensive interpretation of their origin and dynamics. The remarkable velocity of the energy transfer, despite the non-negligible energy separation between the two chromophores, and the analysis of the underlying transport mechanism, highlight the role played by the ground state carotenoid vibrations in assisting the process.

Peptide-based molecular electronic devices are promising due to the large diversity and unique electronic properties of biomolecules. These electronic properties can change considerably with peptide structure, allowing diverse design possibilities. In this work, we explore the effect of the side-chain of the peptide on its electronic properties, by using both experimental and computational tools to detect the electronic energy levels of two model peptides. The peptides include 2Ala and 2Trp as well as their 3-mercaptopropionic acid linker which is used to form monolayers on an Au surface. Specifically, we compare experimental ultraviolet photoemission spectroscopy measurements with density functional theory based computational results. By analyzing differences in frontier energy levels and molecular orbitals between peptides in gas-phase and in a monolayer on gold, we find that the electronic properties of the peptide side-chain are maintained during binding of the peptide to the gold substrate. This indicates that the energy barrier for the peptide electron transport can be tuned by the amino acid compositions, which suggests a route for structural design of peptide-based electronic devices.

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.

Xanthorhodopsin (xR) is a member of the retinal protein family and acts as a proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber. In addition to the retinal chromophore, xR contains a carotenoid, which acts as a light-harvesting antenna as it transfers 40% of the quanta it absorbs to the retinal. Our previous studies have shown that the CD and absorption spectra of xR are dramatically affected due to the protonation of two different residues. It is still unclear whether xR can bind cations. Electron paramagnetic resonance (EPR) spectroscopy used in the present study revealed that xR can bind divalent cations, such as Mn²⁺ and Ca²⁺, to deionized xR (DI-xR). We also demonstrate that xR can bind 1 equiv of Mn²⁺ to a high-affinity binding site followed by binding of ∼40 equiv in cooperative manner and ∼100 equiv of Mn²⁺ that are weakly bound. SQUID magnetic studies suggest that the high cooperative binding of Mn²⁺ cations to xR is due to the formation of Mn²⁺ clusters. Our data demonstrate that Ca²⁺ cations bind to DI-xR with a lower affinity than Mn²⁺, supporting the assumption that binding of Mn²⁺ occurs through cluster formation, because Ca²⁺ cations cannot form clusters in contrast to Mn²⁺.

The barrier energies for isomerization and fragmentation were measured for a series of retinal chromophore derivatives using a tandem ion mobility spectrometry approach. These measurements allow us to quantify the effect of charge delocalization on the rigidity of chromophores. We find that the role of the methyl group on the C13 position is pivotal regarding the ground state dynamics of the chromophore. Additionally, a correlation between quasi-equilibrium isomer distribution and fragmentation pathways is observed.

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.

The dearth of high quality, three dimensional crystals of membrane proteins, suitable for X-ray diffraction analysis, constitutes a serious barrier to progress in structural biology. To address this challenge, we have developed a new crystallization medium that relies on the conjugation of surfactant micelles via base-pairing of complementary hydrophobic nucleosides. Base-pairs formed at the interface between micelles bring them into proximity with each other; and when the conjugated micelles contain a membrane protein, crystal nucleation centers can be stabilized, thereby promoting crystal growth. Accordingly, two hydrophobic nucleoside derivatives deoxyguanosine (G) and deoxycytidine (C), each covalently bonded to a 10 carbon chain were synthesized and added to an aqueous solution containing octyl β-D-thioglucopyranoside micelles. These hydrophobic nucleosides induced the formation of oil-rich globules after 2 days incubation at 19 °C or after a few hours in the presence of ammonium sulfate; however, phase separation was inhibited by 100 mM GMP. The presence of the membrane protein bacteriorhodopsin in the conjugated micellar dispersion resulted in the growth within the colorless globules of a variety of purple crystals, the color indicating a functional protein. On this basis, we suggest that conjugation of micelles via base-pair complementarity may provide significant assistance to the structural determination of integral membrane proteins.

Primary photochemical events in the unusually thermostable proton pumping rhodopsin of Thermus thermophilus bacterium (TR) are reported for the first time. Internal conversion in this protein is shown to be significantly faster than in bacteriorhodopsin (BR), making it the most rapidly isomerizing microbial proton pump known. Internal conversion (IC) dynamics of TR and BR were recorded from room temperature to the verge of thermal denaturation at 70 °C and found to be totally independent of temperature in this range. This included the well documented multiexponential nature of IC in BR, suggesting that assignment of this to ground state structural inhomogeneity needs revision. TR photodynamics were also compared with that of the phylogenetically more similar proton pump Gloeobacter rhodopsin (GR). Despite this similarity GR has poor thermal stability, and the excited state decays significantly more slowly and exhibits very prominent stretched exponential behavior. Coherent torsional wave-packets induced by impulsive photoexcitation of TR and GR show marked resemblance to each other in frequency and amplitude and differ strikingly from similar signatures in pump-probe data of BR and other microbial retinal proteins. Possible correlations between IC rates and thermal stability and the promise of using torsional coherence signatures for understanding chromophore protein binding in microbial retinal proteins are discussed.

Spin-dependent photoluminescence (PL) quenching of CdSe nanoparticles (NPs) has been explored in the hybrid system of CdSe NP purple membrane, wild-type bacteriorhodopsin (bR) thin film on a ferromagnetic (Ni-alloy) substrate. A significant change in the PL intensity from the CdSe NPs has been observed when spin-specific charge transfer occurs between the retinal and the magnetic substrate. This feature completely disappears in a bR apo membrane (wild-type bacteriorhodopsin in which the retinal protein covalent bond was cleaved), a bacteriorhodopsin mutant (D96N), and a bacteriorhodopsin bearing a locked retinal chromophore (isomerization of the crucial C13=C14 retinal double bond was prevented by inserting a ring spanning this bond). The extent of spin-dependent PL quenching of the CdSe NPs depends on the absorption of the retinal, embedded in wild-type bacteriorhodopsin. Our result suggests that spin-dependent charge transfer between the retinal and the substrate controls the PL intensity from the NPs.

Electron transport properties via a photochromic biological photoreceptor have been studied in junctions of monolayer assemblies in solid-state configurations. The photoreceptor studied was a member of the LOV domain protein family with a bound flavin chromophore, and its photochemically inactive mutant due to change of a crucial cysteine residue by a serine. The photochemical properties of the protein were maintained in dry, solid state conditions, indicating that the proteins in the junctions were assembled in native state-like conditions. Significant current magnitudes (>20 μA at 1.0 V applied bias) were observed with a mechanically deposited gold pad (area ∼0.002 cm²) as top electrode. The current magnitudes are ascribed to electrode-cofactor coupling originating from the apparent perpendicular orientation of the protein's cofactor embedded between the electrodes, and its proximity to the electrodes. Temperature independent electron transport across the protein monolayers demonstrated that solid-state electron transport is dominated by tunneling. Modulation of the observed current by illumination of the wildtype protein suggested conformation-dependent electron conduction efficiency across the solid-state protein junctions.

Electron transport (ETp) across met-myoglobin (m-Mb), as measured in a solid-state-like configuration between two electronic contacts, increases by up to 20 fold if Mb is covalently bound to one of the contacts, a Si electrode, in an oriented manner by its hemin (ferric) group, rather than in a non-oriented manner. Oriented binding of Mb is achieved by covalently binding hemin molecules to form a monolayer on the Si electrode, followed by reconstitution with apo-Mb. We found that the ETp temperature dependence (>120 K) of non-oriented m-Mb virtually disappears when bound in an oriented manner by the hemin group. Our results highlight that combining direct chemical coupling of the protein to one of the electrodes with uniform protein orientation strongly improves the efficiency of ET across the protein. We hypothesize that the behavior of reconstituted m-Mb is due to both strong protein-substrate electronic coupling (which is likely greater than in non-oriented m-Mb) and direct access to a highly efficient transport path provided by the hemin group in this configuration.

We observe temperature-independent electron transport, characteristic of tunneling across a ∼6 nm thick Halorhodopsin (phR) monolayer. phR contains both retinal and a carotenoid, bacterioruberin, as cofactors, in a trimeric protein-chromophore complex. This finding is unusual because for conjugated oligo-imine molecular wires a transition from temperature-independent to -dependent electron transport, ETp, was reported at ∼4 nm wire length. In the ∼6 nm long phR, the ∼4 nm 50-carbon conjugated bacterioruberin is bound parallel to the α-helices of the peptide backbone. This places bacterioruberins ends proximal to the two electrodes that contact the protein; thus, coupling to these electrodes may facilitate the activation-less current across the contacts. Oxidation of bacterioruberin eliminates its conjugation, causing the ETp to become temperature dependent (>180 K). Remarkably, even elimination of the retinal-protein covalent bond, with the fully conjugated bacterioruberin still present, leads to temperature-dependent ETp (>180 K). These results suggest that ETp via phR is cooperatively affected by both retinal and bacterioruberin cofactors.

A member of the retinal protein family, halorhodopsin, acts as an inward light-driven Cl^- pump. It was recently demonstrated that the Natronomonas pharaonis halorhodopsin-overproducing mutant strain KM-1 contains, in addition to the retinal chromophore, a lipid soluble chromophore, bacterioruberin, which binds to crevices between adjacent protein subunits. It is established that halorhodopsin has several chloride binding sites, with binding site I, located in the retinal protonated Schiff base vicinity, affecting retinal absorption. However, it remained unclear whether cations also bind to this protein. Our electron paramagnetic resonance spectroscopy examination of cation binding to the halorhodopsin mutant KM-1 reveals that divalent cations like Mn²⁺ and Ca²⁺ bind to the protein. Halorhodopsin has a high affinity for Mn²⁺ ions, which bind initially to several strong binding sites and then to binding sites that exhibit positive cooperativity. The binding behavior is pH-dependent, and its strength is influenced by the nature of counterions. Furthermore, the binding strength of Mn²⁺ ions decreases upon removal of the retinal chromophore from the protein or following bacterioruberin oxidation. Our results also indicate that Mn²⁺ ions, as well as Cl^- ions, first occupy binding sites other than site I. The observed synergetic effect between cation and anion binding suggests that while Cl^- anions bind to halorhodopsin at low concentrations, the occupancy of site I requires a high concentration.

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.

A central vision in molecular electronics is the creation of devices with functional molecular components that may provide unique properties. Proteins are attractive candidates for this purpose, as they have specific physical (optical, electrical) and chemical (selective binding, self-assembly) functions and offer a myriad of possibilities for (bio-)chemical modification. This Progress Report focuses on proteins as potential building components for future bioelectronic devices as they are quite efficient electronic conductors, compared with saturated organic molecules. The report addresses several questions: how general is this behavior; how does protein conduction compare with that of saturated and conjugated molecules; and what mechanisms enable efficient conduction across these large molecules? To answer these questions results of nanometer-scale and macroscopic electronic transport measurements across a range of organic molecules and proteins are compiled and analyzed, from single/few molecules to large molecular ensembles, and the influence of measurement methods on the results is considered. Generalizing, it is found that proteins conduct better than saturated molecules, and somewhat poorer than conjugated molecules. Significantly, the presence of cofactors (redox-active or conjugated) in the protein enhances their conduction, but without an obvious advantage for natural electron transfer proteins. Most likely, the conduction mechanisms are hopping (at higher temperatures) and tunneling (below ca. 150-200 K).

Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. The pH dependence of isomeric composition, dark-adaptation rate, and primary events of Anabaena sensory rhodopsin (ASR), a microbial rhodopsin discovered a decade ago, are presented. The main findings are: (a) Two pK_a values of 6.5 and 4.0 assigned to two different protein residues are observed using spectroscopic titration experiments for both ground-state retinal isomers: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. An additional pK_a value of 8.5 is observed only for 13C-ASR. (b) The isomeric composition of ASR is determined over a wide pH range and found to be almost pH-independent in the dark (>96% AT isomer) but highly pH-dependent in the light-adapted form. (c) The kinetics of dark adaptation is recorded over a wide pH range, showing that the thermal isomerization from 13C to AT retinal occurs much faster at high pH rather than under acidic conditions. (d) Primary photochemical events of ASR at pH 5 are recorded using VIS hyperspectral pump-probe spectroscopy with

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.

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.

The temperature dependence of current-voltage values of electron transport through proteins integrated into a solid-state junction has been investigated. These measurements were performed from 80 up to 400 K [above the denaturation temperature of azurin (Az)] using Si/Az/Au junctions that we have described previously. The current across the similar to 3.5 nm thick Az junction was temperature-independent over the complete range. In marked contrast, for both Zn-substituted and apo-Az (i.e., Cu-depleted Az), thermally activated behavior was observed. These striking temperature-dependence differences are ascribed to the pivotal function of the Cu ion as a redox center in the solid-state electron transport process. Thus, while Cu enabled temperature-independent electron transport, upon its removal the polypeptide was capable only of supporting thermally activated transport.

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.

Excited-state dynamics of xanthorhodopsin (XR) and of salinixanthin (SX) in ethanol were investigated by ultrafast pump-hyperspectral probe spectroscopy. Following excitation to the strongly allowed S₂ state of the SX chromophore, transient spectra were recorded photoselectively in the range 430-850 nm. Global kinetic analysis of these data shows the following. (1) Efficient energy transfer from S₂ of the SX in XR to its retinal moiety is verified here. The lifetime of S₂ in SX is, however, determined to be ∼20 fs, much shorter than previously reported. (2) Branching ratios of excitation transfer from S₂ to Si, to S*, and to retinal in XR are measured leading to species associated difference spectra (SADS) for all the states involved. Strong protein effects are detected on these branching probabilities. (3) Si and S* absorption bands in both systems exhibit anisotropy well below the expected r = 0.4, indicating an angle of ∼25° between the S₀ → S₂ and S₁ →- S_n/S* → S_n transition dipoles. The latter allows confident assignment of the debated S* absorption band to an excited state of SX, and not to "hot" S₀. In light of the extremely fast IC from S₂ to lower excited singlets, possible involvement of ballistic IC in SX, and of coherent energy transfer in XR, are discussed.

The primary photochemical dynamics of Hb. pharaonis Halorhodopsin (pHR) are investigated by femtosecond visible pump-near IR dump-hyperspectral probe spectroscopy. The efficiency of excited state depletion is deduced from transient changes in absorption, recorded with and without stimulated emission pumping (SEP), as a function of the dump delay. The concomitant reduction of photocycle Population is assessed by probing the "K" intermediate difference spectrum. Results show that the cross section for stimulating emission is nearly constant throughout the fluorescent state lifetime. Probing "K" demonstrates that dumping produces a proportionate reduction in photocycle yields. We conclude that, despite its nonexponential internal conversion (IC) kinetics, the fluorescent state in pHR constitutes a single intermediate in the photocycle. This contrasts with Conclusions drawn from the Study of primary events in the related chloride pump from Hb. salinarum (sHR), believed to produce the "K" intermediate from a distinct short-lived subpopulation in the excited state. Our discoveries concerning internal conversion dynamics in pHR are discussed in light of recent expectations for similar excited state dynamics in both proteins.

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.

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.

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.

Isomerization of the 11-cis retinal chromophore in the visual pigment rhodopsin is coupled to motion of transmembrane helix H6 and receptor activation. We present solid-state magic angle spinning NMR measurements of rhodopsin and the metarhodopsin II intermediate that support the proposal that interaction of Trp265^6.48 with the retinal chromophore is responsible for stabilizing an inactive conformation in the dark, and that motion of the β-ionone ring allows Trp265^6.48 and transmembrane helix H6 to adopt active conformations in the light. Two-dimensional dipolar-assisted rotational resonance NMR measurements are made between the C19 and C20-methyl groups of the retinal and uniformly ¹³C-labeled Trp265 ^6.48. The retinal C20-Trp265^6.48 contact present in the dark-state of rhodopsin is lost in metarhodopsin II, and a new contact is formed with the C19 methyl group. We have previously shown that the retinal translates 4-5 Å toward H5 in metarhodopsin II. This motion, in conjunction with the Trp-C19 contact, implies that the Trp265^6.48 side-chain moves significantly upon rhodopsin activation. NMR measurements also show that a packing interaction in rhodopsin between Trp265^6.48 and Gly121 ^3.36 is lost in metarhodopsin II, consistent with H6 motion away from H3. However, a close contact between Gly120^3.35 on H3 and Met86 ^2.53 on H2 is observed in both rhodopsin and metarhodopsin II, suggesting that H3 does not change orientation significantly upon receptor activation.

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.

Spectral modulations induced by 6fsec photoexcitation of bacteriorhodopsin are Fourier analyzed. Long lived undulations are assigned to ground state vibrational coherences, while possible excited state contributions are very short lived consisting mainly of HOOP motions.

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.

Activation of the visual pigment rhodopsin is initiated by isomerization of its retinal chromophore to the all-trans geometry, which drives the conformation of the protein to the active state. We have examined by FTIR spectroscopy the impact of a series of modifications at the ring of retinal on the activation process and on molecular interactions within the binding pocket. Deletion of ring methyl groups at C1 and C5 or replacement of the ring in diethyl or ethyl-methyl acyclic analogues resulted in partial agonists, for which the conformational equilibrium between the Meta I and Meta II photoproduct is shifted from the active Meta II side to the inactive Meta I side. While the Meta II states of these artificial pigments had a conformation similar to those of native Meta II, the Meta I states were different. Modifications on the ring of retinal had a particular impact on the interaction of Glu 122 within the ring-binding pocket and are shown to interfere with the Glu 134-mediated proton uptake during formation of Meta II. We further found, upon partial deletion of ring constituents, a decrease of the entropy change of the transition from Meta I to Meta II by up to 50%, while the concomitant reduction of the enthalpy term was less pronounced. These findings underline the particular importance of the ring and the ring methyl groups and are discussed in a model of receptor activation.

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.

Thermal isomerization of the retinal Schiff base C=N double bond is known to trigger the decay of rhodopsin's Meta I/Meta II photoproduct equilibrium to the inactive Meta III state [Vogel, R., Siebert, F., Mathias, G., Tavan, P., Fan, G., and Sheves, M. (2003) Biochemistry 42, 9863-9874]. Previous studies have indicated that the transition to Meta III does not occur under conditions that strongly favor the active state Meta II but requires a residual amount of Meta I in the initial photoproduct equilibrium. In this study we show that the triggering event, the thermal isomerization of the protonated Schiff base, is independent of the presence of Meta II and occurs even under conditions where the transition to Meta II is completely prevented. We have examined two examples in which the transitions from Lumi to Meta I or from Meta I to Meta II are blocked. This was achieved using dry films of rhodopsin and rhodopsin reconstituted into rather rigid lipid bilayers. In both cases, the resulting fully inactive room temperature photoproducts decay specifically by thermal isomerization of the protonated Schiff base C=N double bond to an all-trans 15-syn chromophore isomer, corresponding to that of Meta III. This thermal isomerization becomes less efficient as the conformation of the respective photoproduct approaches that of Meta II and is fully absent in a pure Meta II state. These results indicate that the decay of the Meta I/Meta II photoproduct equilibrium to Meta III proceeds via Meta I and not via Meta II.

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.

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.

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh_6.10, in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh_6.10 by UV-visible and FTIR spectroscopy and have revealed that Rh_6.10 is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both sub-species relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C₁₃=C₁₄ double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.

New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C₁₃=C₁₄ is required to go between the above structures.

Reaction-induced infrared difference spectra show characteristic amide I spectral changes, which indicate conformational changes of the protein backbone but which cannot be interpreted at a molecular level. To obtain some insights into their causes, we used bacteriorhodopsin as a model system and investigated its BR → N transition during which the largest amide I changes are observed. For the molecular interpretation, we labeled a single peptide C=O group at specific positions of the backbone with ¹³C and monitored the resulting isotope effects. This has been achieved by replacing specific amino acids with a cysteine. Because wild-type bacteriorhodopsin does not contain this amino acid, (1-¹³C)cysteine can be incorporated into the mutants for site-directed isotopic labeling. Although the isotope-induced spectral changes are very small, we observed clear isotope effects for the middle to extracellular part of helices B, C, and F, indicating that the backbone of these parts of the protein is distorted during the reaction, whereas no label effects could be identified for and E-F loop and for the cytosolic regions of helices E and F. The results are discussed within the framework of recent experimental and theoretical studies of the amide I band, and they are correlated to the structural changes observed by other methods.

Absorbance difference spectra were recorded at 20 degreesC from 30 ns to milliseconds after photolysis of lauryl maltoside suspensions of artificial visual pigments derived from 9-cis isomers of 5-ethylretinal, 8,16-methanoretinal (a 6-s-trans-bicyclic analogue), or 5-demethyl-8-methylretinal. In all three pigments, the earliest intermediate that was detected had the characteristics of a mixture of bathorhodopsin and a blue-shifted intermediate, BSI, which is the first decay product of bathorhodopsin in bovine rhodopsin, The first decays resolved on the nanosecond time scale were the formation of the lumirhodopsin analogues. Subsequent decays were able to be fit with a mechanistic scheme which has been shown to apply to both membrane and detergent suspensions of rhodopsin. Large increases were seen in the amount of metarhodopsin I which appeared after photolysis of 5-ethylisorhodopsin and the bicyclic isorhodopsin analogue, while 5-demethyl-8-methylisorhodopsin more closely followed native rhodopsin in decaying through meta I-380, a 380 nm absorbing precursor to metarhodopsin 11, In addition to forming more metarhodopsin 1, the bicyclic analogue stabilized the metarhodopsin I-metarhodopsin 11 equilibrium similarly to what has been previously reported for 9-demethylrhodopsin in detergent, introducing the possibility that the bicyclic analogue could similarly be defective in transducin activation. These observations support the idea that long after initial photolysis, structural details or the retinylidene chromophore continue to play a decisive role in processes leading to the activated form, metarhodopsin II.

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; λ_max = 550 nm) in which isomerization around the critical C₁₃=C₁₄ double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (λ_max = 450 nm) and H430 (λ_max = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.

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.

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. Also described are EPR experiments monitoring reduction and oxidation reactions of a spin label covalently attached to various protein sites. It is concluded that proteins and other macromolecules may undergo structural changes 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.

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 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. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn²⁺ and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn²⁺, which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be ∼9.8± 0.7 Å. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn²⁺ bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.

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.

Hydroretinochromes were reconstituted with aporetinochrome and hydroretinal analogues; 7,8-dihydroretinal, 5,6-dihydroretinal, dehydroretinal, and their analogues. The 'opsin shifts' were 2200 ± 100 cm^-1 for trienals and pentaenals, whereas 1600 ± 100 cm^-1 was the value for tetraenals.

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 hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds: no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pK(a) of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.

During the L → M reaction of the bacteriorhodopsin photocycle the proton of the retinal Schiff base is transferred to the anionic D85. This step, together with the subsequent reprotonation of the Schiff base from D96 in the M → N reaction, results in the translocation of a proton across the membrane. The first of these critical proton transfers occurs in an extended hydrogen-bonded complex containing two negatively charged residues (D85 and D212), two positively charged groups (the Schiff base and R82), and coordinated water. We simplified this region by replacing D212 and R82 with neutral residues, leaving only the proton donor and acceptor as charged groups. The D212N/R82Q mutant shows essentially normal proton transport, but in the photocycle neither of this protein nor of the D212N/R82Q/D96N triple mutant does a deprotonated Schiff base (the M intermediate) accumulate. Instead, the photocycle contains only the K, L, and N intermediates. Infrared difference spectra of D212N/R82Q and D212N/ R82Q/D96N demonstrate that although D96 becomes deprotonated in N, D85 remains unprotonated. On the other hand, M is produced at pH > 8, where according to independent evidence the L ↔ M equilibrium should shift toward M. Likewise, M is restored in the photocycle when the retinal is replaced with the 14-fluoro analogue that lowers the p of the protonated Schiff base, and now D85 becomes protonated as in the wild type. We conclude from these results that the proton transfers to and from the Schiff base probably both occur after photoexcitation of D212N/R82Q, but the L ↔ M and M ↔ N equilibria are shifted away from M, and, untypically, D85 does not retain the proton it had gained. The mechanism of proton transport is not greatly changed when D85 is the only charged component of the Schiff base counterion, but the protonation equilibria in the proton transfer pathway across the protein are drastically altered.

The structure and function of the light-driven proton pump bacteriorhodopsin appear to be determined by the exact geometrical conformation of specific groups in the retinal binding site, including bound water molecules. This applies to the pK_a values of the protonated Schiff base, which links the retinal chromophore to Lys₂₁₆, and to Asp₈₅. In the present work we show that the geometrical constraints imposed by the ring structures of several synthetic retináis can induce substantial changes in the pK_a values of the Schiff base and of Asp₈₅. Thus, the artificial pigments derived from 13-demethyl-11,14-epoxyretinal (2) and 13-demethyl-9,12-epoxyretinal (3) show protonated Schiff base pAa values of 8.2 ± 0.1 and 9.1 ± 0.1, respectively, as compared with 13.3 in the native (all-trans-tetinal) pigment. We also suggest that in both systems the pK_a of Asp₈₅ increases from 3.2 in the native bR to above 9. Analogous, though smaller, effects are obtained for artificial bR pigments derived from 12,14-ethanoretinal (4), 11,-13-propanoretinal (5), 11,13-ethanoretinal (6), and p-(CHR3.)₂N-C₆H₄-HC=CH-C(CH₃)=CH-CHO 7. The effects of geometry on the pK_a values (those on Asp85 being more pronounced) are attributed to the disruption of the original, well-defined, structure in which the Schiff base and its Asp⁸⁵ counterion are bridged by bound water molecules. These results are the first to show that it is possible to modify the pK_a values of the Schiff base and Asp⁸⁵ in appropriate artificial pigments, without inducing intrinsic pAa changes in the chromophore or introducing a mutation in the protein. The results bear on the structure of bR and on the mechanisms of its light-driven proton pump in which both Schiff base and Asp⁸⁵ moieties play central roles.

A system is described that allows for the delineation of the factors that effect the complexation of retinal to the apoprotein of bacteriorhodopsin. This complexation is investigated in various states of hydration, in H₂O and D₂O, at a variety of pH levels, with mutant membranes and labeled retinals. The complexation reaction was also investigated using absorption spectroscopy and vibrational spectra using difference Fourier transform infrared spectroscopy. The results demonstrate the crucial role of water in controlling the protein conformations that lead to protein/ligand binding reactions and begin to shed new light on the protein control of a reaction that normally cannot take place in an aqueous medium.

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.

Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K216. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Zimányi, L., Váró, G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-8543] we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L ⟺ M₁ ⟺ M₂ + H⁺ (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various pH values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L ⟺ M₁ reaction and represents the redistribution of the proton between the Schiff base and D85. From the structure of the protein, the V49A and V49M residue replacements were both predicted to cause decreased alignment of the Schiff base and D85, and indeed we found that they both changed the equilibrium toward the protonated Schiff base. In contrast, the residue replacements A53V and A53G were predicted to move the Schiff base in opposite directions, away from and closer to alignment with D85, respectively. The former indeed changed the equilibrium toward the protonated Schiff base and the latter toward the deprotonated Schiff base. In addition, the hydroxyl stretch band of a bound water in the L state was affected by all mutations that disfavor proton transfer to D85. We conclude that the geometry of the proton donor and acceptor in the Schiff base-D85 pair, mediated by bound water, is a determinant of the proton transfer equilibrium.

The photoreactions of rhodopsin regenerated with three 9-cis retinal analogs, modified at or in the vicinity of the β-ionone ring (namely 5,6-epoxy, 7,8-diH, diethyl-acyclic) have been investigated by UV-vis and FTIR difference spectroscopy. In parallel, the ability to catalyze the GDP → GTP exchange of G-protein (transducin) has been monitored by time-dependent fluorescence spectroscopy. The first photoproduct obtained with all three pigments at liquid nitrogen temperature is a blue-shifted intermediate (BSI), followed by a lumi-like intermediate at 170 K. For the 5,6-epoxy-ISO and 7,8-diH-ISO pigment we obtain two further intermediates similar to the META-I and META-II states of native RHO. For the diethyl-acyclic-ISO pigment only one further intermediate can be stabilized at 280 K. As compared to META-II the respective photoproduct exhibits striking differences. The latter two pigments have also been investigated in the solubilized lipid-free state (detergent: dodecyl maltoside) at 280 K. For the 5,6-epoxy-ISO pigment, the UV-vis, FTIR, and activation data agree with the formation of a META-II-like photoproduct (81% activation). Less META-II formation is observed for the 7,8-dihydro-ISO pigment in membranes (65% activation), but full formation in detergent (100% activation). Neither the membrane-bound nor the solubilized diethyl-acyclic-ISO pigment forms a META-II-like intermediate (18% and 0% activation, respectively). Therefore, we conclude that the substitution of the β-ionone ring by two ethyl groups abolishes steric interactions with the protein, which are essential for META-II formation. The UV-vis and FTIR spectroscopic data are discussed in connection with the biochemical data and the molecular events are interpreted in terms of chromophore-protein interaction.

Bacteriorhodopsin pigments lacking the retinal-Lys-216 covalent bond were prepared by reconstituting the K216G mutant protein with retinal alkylamine Schiff bases. The procedure follows the approach of Zhukovsky et al. [Zhukovsky, E., Robinson, P., & Oprian, D. (1991) Science 251, 558-560] in the case of visual (rhodopsin) pigments. Reconstitution leads to a mixture of three pigments. One of them, bR(K216G)/566a, absorbs (pH = 6.9) at 566 nm. Its absorption is pH-dependent, exhibiting a purple to blue transition. The pigment's laser-induced photocycle patterns are similar to those of wild-type all-trans-bR. A second component, bR(K216G)/566b, exhibits an independent photocycle reminiscent of that of wild-type 13-cis-bR. A third pigment component, bR(K216G)/630, absorbs around 630 nm. Experiments in the presence of a pH dye indicator show that illumination of bR(K216G)/566 produces a detectable proton gradient. It is concluded that a covalent bond between the retinal chromophore and the protein backbone is not a prerequisite for the basic structure and photochemical features of bR or for its proton pump activity.

Results are presented demonstrating that the backbone of the active site lysine of bacteriorhodopsin undergoes light-induced structural alterations during bacteriorhodopsin-mediated light-induced proton pumping. This conclusion is based on difference Fourier transform infrared spectroscopy of isotopically labeled bacteriorhodopsin. The data demonstrate that the backbone carbonyl of lysine achieves an extremely low vibrational frequency during M(412) intermediate formation. This is preceded by a structural transition in the lysine backbone that leads to an active site lysine carbonyl with the observed low vibrational frequency, probably due to a high degree of solvation.

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 active site of an ion pump must communicate alternately with the two opposite membrane surfaces. In the light-driven proton pump, bacteriorhodopsin, the retinal Schiff base is first the proton donor to D85 (with access to the extracellular side), and then it becomes the acceptor of the proton of D96 (with access to the cytoplasmic side). This "reprotonation switch" has been associated with a protein conformation change observed during the photocycle. When D85 is replaced with asparagine, the pK_R value of the Schiff base is lowered from above 13 to about 9. We determined the direction of the loss or gain of the Schiff base protin in unphotolyzed and in photoexcited D85N, and the D85N/D96N and D85N/D96A double mutants, in order to understand the intrinsic and the induced connectivities of the Schiff base to the two membrane surfaces. The influence of D96 mutations on proton exchange and on acceleration of proton shuttling to the surface by azide indicated that in either case the access of the Schiff base on D85N mutants is to the cytoplasmic side. In the wild-type protein (but with the pK_a of the Schiff base lowered by 13-trifluoromethyl retinal substitution) the results suggested that the Schiff base can communicate also with the extracellular side. Raising the pH without illumination of D85N so as to deprotonate the Schiff base caused the same, or nearly the same, change of X-ray scattering as observed when the Schiff base deprotonates during the wild-type photocycle. The results link the charge state of the active site of the global protein conformation and to the connectivity of the Schiff base proton to the membrane surfaces. Their relationship suggests that the conformation of the unphotolyzed wild-type protein is stabilized by coulombic interaction of the Schiff base with its counter-ion. A proton is translocated across the membrane after light-induced transfer of the Schiff base proton to D85, because the protein assumes an alternative conformation that separates the donor from the acceptor and opens new conduction pathways between the active site and the two membrane surfaces.

The Schiff base linkage bond configuration of bacteriorhodopsin was studied using model compounds consisting of all-trans- and 13-cis-retinal-protonated Schiff bases bearing CN anti and syn bond configurations. The CN configuration was analyzed using a combination of Fourier transform infrared spectroscopy and isotopically labeled chromophores. It was found that, in the model compounds, the coupling between the C₁₄C₁₅ stretching frequency and the NH rock is weak in the all-trans-retinal-protonated Schiff base in both the anti and syn CN configurations. However, this coupling is relatively strong in the 13-cis-retinal-protonated Schiff base in both the anti and syn CN configurations. Thus, it is concluded that, in model compounds, the C₁₄C₁₅ mode can serve as a marker for the C₁₃C₁₄ bond configuration but not for the CN. A different situation may prevail in bacteriorhodopsin due to different conformations of the retinal chromophore in the protein binding site and in solution. This difference suggests that the C₁₄C₁₅/NH coupling in retinal-protonated Schiff bases is affected by the retinal conformation.

The resonance Raman (RR) spectrum of a K-intermediate formed during the photoreaction of an artificial bacteriorhodopsin (BR) pigment (derived from E-11,20 ethanoretinal) containing a six-membered ring spanning the C₁₁=C₁₂-C₁₃ region of the retinal chromophore (BR6.11) is recorded by picosecond time-resolved resonance Raman (PTR³) spectroscopy. A recent study of the BR6.11 photoreaction utilizing picosecond transient absorption and picosecond time-resolved fluorescence measurements revealed that (i) a J-intermediate (J6.11) appears within

Artificial bovine rhodopsin pigments derived from synthetic retinal analogues carrying electron-withdrawing substituents (fluorine and chlorine) were prepared. The effects of the electron withdrawing substituents on the pKa values of the pigments and on the corresponding Schiff bases in solution were analyzed. The data suggest that the apparent pKa of the protonated Schiff base is above 16. However, the alternative possibility that the retinal Schiff base linkage in bovine rhodopsin is not accessible for titration from the aqueous bulk medium cannot be definitely ruled out.

Absorption maxima and C-13 NMR chemical shifts were measured for retinal iminium salts. A good correlation was found between the absorption maxima and the C-13 chemical shifts of the retinal polyene carbons within the same solvent system. The chemical shifts of the odd-membered carbons of the polyene are affected much more than the even carbons by pi-electron delocalization caused by perturbations in the Schiff base linkage vicinity. The absorption maxima of bacteriorhodopsin (bR) (568 nm) is closely mimicked by protonated Schiff base chromophores bearing ring-chain s-trans planarity and weak hydrogen bonding between their positively charged Schiff base linkage and its counteranion. These chromophores exhibit a C5 chemical shift similar to the unusual one found in bacteriorhodopsin. These results indicate that it is possible to closely mimic the absorption maximum of bR and its C5 chemical shift without requiring a nonconjugated negative charge in the vicinity of the retinal ring. The effect of nonconjugated positive and negative charges on the C-13 chemical shifts of the retinal polyene is evaluated using synthetic retinal chromophores. The charges affect the chemical shift in an alternating fashion (namely, upfield and downfield shifts) and mainly affect the double bond located in the immediate vicinity of the charge. The influence of the charge is diminished as its distance is increased. The spatial arrangement of the charge, relative to the polyene, is crucial for its effect. A symmetric C = C/charge arrangement causes only a minor change in the chemical shift, still, however, affecting the absorption maximum of the retinal protonated Schiff base. The implications of these measurements for bacteriorhodopsin are discussed.

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.

In variance with chlorophyll-based photosynthetic pigments, the triplet states of rhodopsins, either visual or photosynthetic, have not been observed experimentally. This is due to the ultrafast crossing from S1 to S0, which effectively competes with intersystem crossing to the triplet (T1) state. In order to populate T1 indirectly, laser photolysis experiments are performed with model protonated Schiff bases of retinal in solution, in which both inter- and intramolecular energy transfer to the polyene chromophore are carried out from an appropriate triplet energy donor. The experiments are then extended to bacteriorhodopsin (bR) by detaching the native retinal chromophore from the protein-binding site and replacing it by an analogous (synthetic) protonated Schiff base polyene, attached in a nonconjugated way to a naphthone triplet donor. Pulsed laser excitation of the latter moiety led, for the first time, to the observation of the triplet state of a rhodopsin. Possible locations and roles of the T1 state in bR and in visual pigments are discussed briefly.

Nanosecond time-resolved and continuous illumination, low-temperature, spectroscopic studies reveal a new photolysis intermediate in a wide variety of artificial visual pigments as well as in native rhodopsin. This new intermediate, BSI, has a blue-shifted spectrum relative to the pigments as well as to their batho and lumi intermediates. At room temperature BSI is formed subsequent to batho and approaches an equilibrium with batho before decaying to the lumi intermediate. Chromophore modifications, which modify the j8-ionone ring, eliminate conjugation between the ring and the polyene chain, add bulky groups to the C4 position on the ring, or remove the 13-methyl group all yield time-resolved spectra which lead to the general scheme [equation omitted] The same mechanism is shown to be valid in isorhodopsin and in the native bovine pigment rhodopsin. Chromophore modifications described above affect the batho s=t BSI equilibrium as well as the kinetics of approach to equilibrium but have little effect on the spectra of the intermediates or on the rate of the BSI to lumi transition. Implications for the nature of the BSI intermediate are discussed. Though BSI has a spectrum blue-shifted from that of batho, BSI is higher in enthalpy. It is proposed that this apparent conflict may be due to the fact that the photon energy, initially stored in chromophore-protein interactions, is transmitted to the protein during the batho-to-BSI transition. If energy at the BSI stage is still stored in the chromophore, models simply relating energy storage to bathochromic shifts must be ruled out.

The surface potential of the purple membrane was measured by a novel method by using an artificial bacteriorhodopsin whose chromophore was 13-CF₃ retinal instead of retinal. When attached to the apoprotein by a Schiff base, the intrinsic pK of the 13-CF₃ chromophore is around 7.3. The apparent p_K of this pigment depends on the surface potential and thus on the electrolyte concentration. This allowed us to determine the surface charge density using the Gouy-Chapman equation. The surface charge density was found to be -1.65 ± 0.15 × 10^-3 electronic charges per Ų or about 2 negative charges/bacteriorhodopsin. This large value for the surface potential probably explains both part of the strong apparent association of divalent cations with the membrane and the effect of low salt concentrations on light-induced proton release from the purple membrane.

The photolysis intermediates of an artificial bovine rhodopsin pigment, cis-5,6-dihydro-isorhodopsin (cis-5,6,-diH-ISORHO, lambda max 461 nm), which contains a cis-5,6-dihydro-9-cis-retinal chromophore, are investigated by room temperature, nanosecond laser photolysis, and low temperature irradiation studies. The observations are discussed both in terms of low temperature experiments of Yoshizawa and co-workers on trans-5,6-diH-ISORHO (Yoshizawa, T., Y. Shichida, and S. Matuoka. 1984. Vision Res. 24: 14551463), and in relation to the photolysis intermediates of native bovine rhodopsin (RHO). It is suggested that in 5,6-diH-ISORHO, a primary bathorhodopsin intermediate analogous to the bathorhodopsin intermediate (BATHO) of the native pigment, rapidly converts to a blue-shifted intermediate (BSI, lambda max 430 nm) which is not observed after photolysis of native rhodopsin. The analogs from lumirhodopsin (LUMI) to meta-II rhodopsin (META-II) are generated subsequent to BSI, similar to their generation from BATHO in the native pigment. It is proposed that the retinal chromophore in the bathorhodopsin stage of 5,6-diH-ISORHO is relieved of strain induced by the primary cis to trans isomerization by undergoing a geometrical rearrangement of the retinal. Such a rearrangement, which leads to BSI, would not take place so rapidly in the native pigment due to ring-protein interactions. In the native pigment, the strain in BATHO would be relieved only on a longer time scale, via a process with a rate determined by protein relaxation.

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.

Do the absorption maxima of the cyanines 13 shift in dependence of the position of the counterions? In C₂H₅OH and CH₂Cl₂ no such interaction could be established. This finding should contribute, inter alia, to an understanding of the role of the natural bacteriorhodopsins. (Figure Presented.)

Artificial bacteriorhodopsin pigments based on synthetic retinal analogues carrying an electron-withdrawing CF₃ substituent group were prepared. The effects of CF₃ on the spectra, photocycles, and Schiff base pK(a) values of the pigments were analyzed. A reduction of 5 units in the pK(a) of the Schiff base is observed when the CF₃ substituent is located at the C-13 polyene position, in the vicinity of the protonated Schiff base nitrogen. The results lead (i) to the unambiguous characterization of the (direct) titration of the Schiff base in bacteriorhodopsin and (ii) to the conclusion that the deprotonation rate of the Schiff base during the photocycle (i.e., the generation of the M₄₁₂ intermediate) is determined by a structural change in the protein.

A series of modified retinals bearing nonconjugated positive charges along the polyene were synthesized. It was found that nonconjugated charges shifted the absorption maxima of retinal chromophore as well as protonated retinal Schitf bases. The magnitude of shift observed in bacteriorhodopsin (bR), 5170 cm^-1, could be found in our models by the additivity of two factors: (1) interaction through space with a positive charge located in the vicinity of the ring operating in nonprotic solvents, provided that the interaction between the charge and its counteranion is weakened by a homoconjugation effect; (2) weakening the interaction of the Schiff base positively charged nitrogen with its counteranion. A shift of ca. 5000 cm^-1 can also be achieved by interaction through space with two nonconjugated positive charges. The absorption maximum of protonated retinal Schiff base is influenced significantly by an interaction with a nonconjugated charge located in the vicinity of the ring moiety or carbon 9. The influence of a charge located in the vicinity of carbon 12 and carbon 14 is minor. Nonconjugated positive charges have a remarkable effect on C=C stretching frequencies as well. It is suggested that the different C=C stretching frequencies found in bR, visual pigments, as well as their photochemically induced intermediates, may originate from interaction with external charges, the C=N⁺ stretching frequency does no exhibit similar sensitivity to external nonconjugated charges, and it is practically unaffected by them.

Artificial bacteriorhodopsin (bR) pigments based on synthetic retinal analogues with selectively blocked single and double bonds are prepared and submitted to pulsed laser photolysis. Similar experiments are carried out with short-chain aromatic analogues. It is concluded that only the C13=C14 double bond can be isomerized in the primary photoprocess. It is shown also that this process is accompanied by separation of the Schiff base from its protein counterion. The effective dielectric constant at the binding site and the nature of the Schiff base counterion play an important role in determining the absorption maximum of bR.

Linear dichroism spectra of several retinoids and related polyenes incorporated in stretched polyethylene films were determined. It is suggested that the retinoids are oriented with the plane of the ring parallel to the stretching direction of the film, the long polyene chain being displaced from that direction.

Abstract Three artificial bacteriorhodopsins are prepared [from synthetic aromatic and bicyclic analogues of retinal and exposed to spectroscopic and pulsed lader photolysis studies. The spectra of the pigments, all perturbed in the ring region of the molecule, are markedly blue shifted in respect to natural bacteriorhodopsin. The shift is attributed to a decreased effect of a protein charge in the vicinity of the ring, in agreement with the pointcharge model of Nakanishi et al., 1980. The photocycles of the synthetic pigments exhibit a primary redshifted (K) intermediate and a blue shifted (M) transient, analogous to those observed for the natural pigment. Such observations impose considerable limitations, both on the possible chromophore conformational changes and on the effects of neighbouring protein charges associated with the photocycle. It is concluded that only the Schiff base counterion, but not the ring charge, may be associated with the generation of the primary red shifted K species. Moreover, the rigidity imposed on the polyene by the additional ring in the bicyclic analogue shows that the photocycle can not be initiated by conformational changes in the retinyl moiety up to the C₉ carbon in the polyene chain. It is also observed that the K→L process in the photocycle is considerably slower in the case of the synthetic pigments. The observation is rationalized by attributing the process to a conformational change in the polyene moiety catalyzed by the ring protein charge.

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.

This paper presents our attempts to utilize polarized (linear dichroic) spectroscopy of compounds oriented in stretched films for the conformational analysis of labile systems.

1α-Hydroxy[7-³H]cholecalciferol (specific radioactivity of 2Ci/mmol) was synthesized, and its metabolism in chicks studied. 2. 1α-Hydroxy[7-³H]cholecalciferol was metabolized very rapidly in the chick to 1α,25-dihydroxy[7-³H]cholecalciferol and to a metabolite less polar than 1α-hydroxycholecalciferol. Intestine exhibited highest accumulation of 1α-25-dihydroxy[7-³H]cholecalciferol, and liver exhibited highest accumulation of the non-polar metabolite. Tissue uptake of 1α-hydroxy[7-³H]cholecalciferol and its metabolites in chicks that were dosed continuously for 16 days with 1α-hydroxy[7-³H]cholecalciferol did not exceed by very much that observed in tissues obtained from chicks that were dosed with a single injection of 1α-hydroxy[7-³H]cholecalciferol 24h before killing, except for liver and kidney. Lowest accumulation of metabolites was noted in muscle and bone, and for the latter, highest uptake of 1α,25-dihydroxy[7-³H]cholecalciferol was noted in the epiphysial periosteum and the metaphysis. Formation of 1α,24,25-trihydroxy[7-³H]cholecalciferol was not observed in the chicks that were dosed continuously with 1α-hydroxy[7-³H]cholecalciferol, despite the fact that plasma calcium and phosphorus were normal and despite the presence of renal 24-hydroxylase activity. The vitamin D status of the chicks did not appear to affect the metabolic profile of the administered 1α-hydroxy[7-³H]cholecalciferol.

(6R)-Hydroxy-3,5-cyclovitamin D₃ was converted with HF, HCl, and HBr into 3β-fluoro-, 3β-chloro-, and 3β-bromo-3-deoxyvitamin D₃ respectively, and with NaI-ZnCl₂ into the corresponding 3β-iodo derivative; a 3β-fluoro-1α-hydroxyvitamin D₃ analogue was prepared from 1α-hydroxyvitamin D₃ tosylate using the 3,5-cyclovitamin derivative as an intermediate.

1 β-Hydroxyvitamin D₃ was prepared from 1α-hydroxyvitamin D₃ by oxidation to 1-ketoprevitamin D₃, followed by sodium borohydride reduction and subsequent thermal isomerization. The conformational equilibria in 1β-hydroxyvitamin D₃ were established using ¹H NMR technique. These data indicate that in nonpolar solvent this compound assumes mainly the conformation in which hydroxy groups are both axial, while in H-bonding solvent mainly the conformation where these groups are both diequatorial.

6-Methylvitamin D₃ and its isomers, the corresponding previtamin, trans-vitamin, and tachysterol were prepared from 6-oxo-3,5-cyclovitamin D₃; their relative thermodynamic stabilities were established, and compared with the stabilities of the respective vitamin D₃ isomers.