Publications

The term supramolecular polymer has been applied to polymeric materials in which the individual units, i.e., building blocksare bound to each other via noncovalent interactions, including electrostatic or hydrogen bonding, as well as metalligand conjugation. The building blocks are generally low molecular weight amphiphiles. Methods for preparing biopolymers based on non-toxic, metalligand conjugation have been little studied; however, they offer significant potential for tuning the response of biologically relevant macromolecules. In this communication, we characterize the assembly and morphology of supramolecular biopolymers in which the building blocks are low- or medium-molecular weight globular proteinsubiquitin and Cas9-interacting via metalligand conjugation. In each case, the protein gene was expressed in cell culture with the addition of hexa-His/linkers at both the N and C termini. Divalent cations investigated were Zn²⁺ and Ni²⁺. We observe in cryo-TEM imaging an absolute requirement for divalent cations for the formation of supramolecular biopolymers. In the presence of Ni²⁺, 1D assembled fibers are predominant, while with Zn²⁺, the more frequently detected structures are sheet-like. We use gel electrophoresis and CD spectroscopy to monitor possible secondary and tertiary structural changes in the protein building blocks during conjugation.

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.

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

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

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.

The protonated Schiff-base retinal acts as the chromophore in bacteriorhodopsin as well as in rhodopsin. In both cases, photoexcitation initializes fast isomerization which eventually results in storage of chemical energy or signaling. The details of the photophysics for this important chromophore is still not fully understood. In this study, action-absorption spectra and photoisomerization dynamics of three retinal derivatives are measured in the gas phase and compared to that of the protonated Schiff-base retinal. The retinal derivatives include C₉=C₁₀trans-locked, C₁₃=C₁₄trans-locked and a retinal derivative without the β-ionone ring. The spectroscopy as well as the isomerization speed of the chromophores are altered significantly as a consequence of the steric constraints.

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.

Bacteriorhodopsin (bR), a proton-pumping protein that converts light into electrochemical energy, has recently attracted attention for solar energy conversion applications. However, its application for electrochemical supercapacitors has not been reported due to the demanding requirements on ordered oriented membranes. Herein, the authors explore bR for supercapacitor application by decorating oriented bR-monolayers onto the surface of an ordered and highly capacitive polyaniline (PANI)-nanofilm prepared by electropolymerization on an Au electrode. The prepared PANI-nanofilm displays good and tunable pseudocapacitive performance attributed to its multiple proton doping states. Due to the synergistic contribution of light-induced proton pumping capacity and the photoelectric function of bR, the specific capacitance of the bR/PANI/Au-electrode has been enhanced pronouncedly by the introduction of oriented bR-monolayers, and has been further improved upon light irradiation. At a current density of 60 A g(-1), a specific capacitance as high as 1146 F g(-1) was achieved following 550 nm light irradiation for the as-prepared bR/PANI-nanocomposites.

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.

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

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.

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

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.

Electron transfer (ET) proteins are biomolecules with specific functions, selected by evolution. As such they are attractive candidates for use in potential bioelectronic devices. The blue copper protein azurin (Az) is one of the most-studied ET proteins. Traditional spectroscopic, electrochemical, and kinetic methods employed for studying ET to/from the protein's Cu ion have been complemented more recently by studies of electrical conduction through a monolayer of Az in the solid-state, sandwiched between electrodes. As the latter type of measurement does not require involvement of a redox process, it also allows monitoring electronic transport (ETp) via redox-inactive Az-derivatives. Here, results of macroscopic ETp via redox-active and -inactive Az derivatives, i.e., Cu(II) and Cu(I)-Az, apo-Az, Co(II)-Az, Ni(II)-Az, and Zn(II)-Az are reported and compared. It is found that earlier reported temperature independence of ETp via Cu(II)-Az (from 20 K until denaturation) is unique, as ETp via all other derivatives is thermally activated at temperatures >≈200 K. Conduction via Cu(I)-Az shows unexpected temperature dependence >≈200 K, with currents decreasing at positive and increasing at negative bias. Taking all the data together we find a clear compensation effect of Az conduction around the Az denaturation temperature. This compensation can be understood by viewing the Az binding site as an electron trap, unless occupied by Cu(II), as in the native protein, with conduction of the native protein setting the upper transport efficiency limit.

The retinal proton pump xanthorhodopsin (XR) was recently found to function with an attached carotenoid light harvesting antenna, salinixanthin (SX). It is intriguing to discover if this departure from single chromophore architecture is singular or if it has been adopted by other microbial rhodopsins. In search of other cases, retinal protein encoding genes in numerous bacteria have been identified containing sequences corresponding to carotenoid binding sites like that in XR. Gloeobacter rhodopsin (GR), exhibiting particularly close homology to XR, has been shown to attach SX, and fluorescence measurements suggest SX can function as a light harvesting (LH) antenna in GR as well. In this study, we test this suggestion in real time using ultrafast transient absorption. Results show that energy transfer indeed occurs from S₂ of SX to retinal in the GR-SX composite with an efficiency of ∼40%, even higher than that in XR. This validates the earlier fluorescence study, and supports the notion that many microbial retinal proteins use carotenoid antennae to harvest light.

Xanthorhodopsin (xR) is a retinal protein that contains, in addition to the retinal moiety, a salinixanthin chromophore absorbing at 456, 486, and 520 nm [ Balashov, S. "P.; Science 2005, 309, 2061 ]. The CD spectrum of xR is very unique with a "conservative" character, containing negative and positive lobes and resembling the first derivative of the absorption spectrum [ Balashov, S. P.; Biochemistry 2006, 45, 10998 ]. It was suggested that the CD spectrum is likely to be composed of several components and that the salinixanthin interacts closely with the retinal chromophore [ Balashov, S. P.; Biochemistry 2006, 45, 10998; Imasheva, E. S.; Photochem. Photobiol. 2008, 84, 977; Lanyi, J. K.; Acta Bioenerg. 2008, 1777, 684; Smolensky, E.; Biochemistry 2009, 48, 8179; Smolensky Koganov, E.; Biochemistry 2013, 52, 1290 ]. In this work, we aim to further explore the nature and origin of the unique CD spectrum of xR. We follow the absorption and CD spectra at different pHs of wild-type (wt) xR and of artificial xR pigments, characterized by a shifted absorption maximum of the retinal chromophore, as well as their corresponding reduced retinal protonated Schiff base pigments. Our results revealed a protein residue (other than the protonated Schiff base counterion), for which protonation affects the CD spectrum by decreasing the negative lobe at ∼530 nm and the positive lobes at 478 and 455 nm, which might be due to elimination of excitonic coupling between the salinixanthin chromophores, although other possibilities cannot be completely excluded. This spectrum change occurs by the pH decreasing, even in artificial pigment where the absorption of the retinal pigment is significantly shifted from 570 to about 450 nm. The possible excitonic coupling between the salinixanthin chromophores and its contribution to the CD spectrum of xR were supported by a good fitting of the CD spectrum to conservative (excitonic) bands [ Zsila, F.; Tetrahedron: Asymmetry 2001, 12, 3125; Zsila, F.; Tetrahedron: Asymmetry 2002, 13, 273 ]. We propose that the CD spectrum of xR consists of contributions from an excitonic coupling interaction between the salinixanthins chromophores located in different subunits of the 3D structure of xR, the chiral conformation of the salinixanthin within its binding site, and the contribution of the retinal chromophore to the negative lobe at around 550 nm.

GR is directly shown by ultrafast pump-probe measurements to bind the carotenoid Salinixanthin, which acts as an efficient light harvesting antenna. Along with Xanthorhodopsin, This proves light harvesting to be a prevalent strategy in retinal proteins.

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

Integrating proteins in molecular electronic devices requires control over their solid-state electronic transport behavior. Unlike "traditional" electron transfer (ET) measurements of proteins that involve liquid environments and a redox cycle, no redox cofactor is needed for solid-state electron transport (ETp) across the protein. Here we show the fundamental difference between these two approaches by macroscopic area measurements, which allow measuring ETp temperature dependence down to cryogenic temperatures, via cytochrome C (Cyt C), an ET protein with a heme (Fe-porphyrin) prosthetic group as a redox centre. We compare the ETp to electrochemical ET measurements, and do so also for the protein without the Fe (with metal-free porphyrin) and without porphyrin. As removing the porphyrin irreversibly alters the protein's conformation, we repeat these measurements with human serum albumin (HSA), 'doped' (by non-covalent binding) with a single hemin equivalent, i.e., these natural and artificial proteins share a common prosthetic group. ETp via Cyt C and HSA-hemin are very similar in terms of current magnitude and temperature dependence, which suggests similar ETp mechanisms via these two systems, thermally activated hopping (with ∼0.1 eV activation energy) >190 K and tunneling by superexchange

Electron-transfer (ET) rates are measured by use of time-resolved EPR spectroscopy, involving photooxidation of nitroxyl radicals by a ruthenium bipyridyl complex. This permits acquisition of the fundamental characteristics of ET in solution. The method was used on two spin-labeled derivatives of bacteriorhodopsin, and is applicable to proteins, nucleic acids, and biological membranes.

Monolayers of the redox protein Cytochrome C (CytC) can be electrostatically formed on an H-terminated Si substrate, if the protein- and Si-surface are prepared so as to carry opposite charges. With such monolayers we study electron transport (ETp) via CytC, using a solid-state approach with macroscopic electrodes. We have revealed that currents via holo-CytC are almost 3 orders of magnitude higher than via the heme-depleted protein (→ apo-CytC). This large difference in currents is attributed to loss of the proteins' secondary structure upon heme removal. While removal of only the Fe ion (→ porphyrin-CytC) does not significantly change the currents via this protein at room temperature, the 30-335 K temperature dependence suggests opening of a new ETp pathway, which dominates at high temperatures (>285 K). These results suggest that the cofactor plays a major role in determining the ETp pathway(s) within CytC.

Xanthorhodopsin (xR) is a retinal protein that contains, in addition to the retinal chromophore, a carotenoid (salinixanthin) that functions as a light-harvesting antenna [Balashov, S. P., et al. (2005) Science 309, 2061-2064]. The center-center distance between the two polyene chains is 12-13 Å, but the distance between the two rings of retinal and salinixanthin is surprisingly small (∼5 Å) with an angle of ∼45 [Luecke, H., et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 16561-16565]. We aimed to clarify the role of the β-ionone ring in the binding of retinal to apo-xR, as well as a possible role that the β-ionone ring plays in fixation of the salinixanthin 4-keto ring. The binding of native retinal and series of synthetic retinal analogues modified in the β-ionone ring to apo-xR was monitored by absorption and circular dichroism (CD) spectroscopies. The results indicate that the β-ionone ring modification significantly affected formation of the retinal-protein covalent bond as well as the pigment absorption and CD spectra. It was observed that several retinal analogues, modified in the retinal β-ionone ring, did not bind to apo-xR and did not form the pigment. Also, none of these analogues induced the fixation of the salinixanthin 4-keto ring. In addition, we show that the native retinal within its binding site adopts exclusively the 6-s-trans ring-chain conformation.

Measuring solid-state electron transport (ETp) across proteins allows studying electron transfer (ET) mechanism(s), while minimizing solvation effects on the process. ETp is, however, sensitive to any static (conformational) or dynamic (vibrational) changes in the protein. Our macroscopic measurements allow extending ETp studies to low temperatures, with the concomitant resolution of lower current densities, because of the larger electrode contact areas. Thus, earlier we reported temperature-independent ETp via the copper protein azurin (Az), from 80 K until denaturation, whereas for apo-Az ETp was temperature dependent above 180 K. Deuteration (H/D substitution) may provide mechanistic information on the question of whether the ETp involves H-bonds in the solid state. Here we report results of kinetic deuterium isotope effect (KIE) measurements on ETp through holo-Az as a function of temperature (30-340 K). Strikingly, deuteration changed ETp from temperature independent to temperature dependent above 180 K. This H/D effect is expressed in KIE values between 1.8 (340 K) and 9.1 (≤180 K). These values are remarkable in light of the previously reported inverse KIE on ET in Az in solution. We ascribe the difference between our KIE results and those observed in solution to the dominance of solvent effects in the latter (larger thermal expansion in H ₂O than in D₂O), whereas in our case the KIE is primarily due to intramolecular changes, mainly in the low-frequency structural modes of the protein caused by H/D exchange. The observed high KIE values are consistent with a transport mechanism that involves through-H-bonds of the β-sheet structure of Az, likely also those in the Cu coordination sphere.

Solid-state electron transport (ETp) via a monolayer of immobilized azurin (Az) was examined by conducting probe atomic force microscopy (CP-AFM), as a function of both temperature (248-373K) and applied tip force (6-15 nN). At low forces, ETp via holo-Az (with Cu²⁺) is temperature-independent, but thermally activated via the Cu-depleted form of Az, apo-Az. While this observation agrees with those of macroscopic-scale measurements, we find that for holo-Az the mechanism of ETp at high temperatures changes upon an increase in the force applied by the tip to the proteins; namely, above 310 K and forces >6 nN ETp becomes thermally activated. This is in contrast to apo-Az, where increasing applied force causes only small monotonic increases in currents due to decreased electrode separation. The distinct ETp temperature dependence of holo- and apo-Az is assigned to a difference in structural response to pressure between the two protein forms. An important implication of these CP-AFM results (of measurements over a significant temperature range) is that for reliable ETp measurements on flexible macromolecules, such as proteins, the pressure applied during the measurements should be controlled or at least monitored.

Femtosecond spectroscopy is used to compare photochemical dynamics in light-adapted and dark-adapted bacteriorhodopsin (BR). The retinal prosthetic group is initially all-trans in the former, while it is nearly a 1:1 mixture with 13-cis in the latter. Comparing photochemistry in both serves to assess how the initial retinal configuration influences internal conversion and photoisomerization dynamics. Contrary to an earlier study, our results show that after excitation of the 13-cis form it crosses back to the ground state much more rapidly than the biologically active all-trans reactant. A similar result was recently obtained for another microbial retinal protein, Anabaena Sensory Rhodospin (ASR), which can be toggled by light between two analogous ground state configurations. Together, these studies suggest that this disparity in rates may be a general trend in the photochemistry of microbial retinal proteins. This may bear as well on the well-known enhancement in photoisomerization rates going from microbial retinal proteins to the visual pigments, as the latter also start the course of photoreception in a cis retinal configuration, in that case 11-cis. In lieu of indications for pretwisting or straining of the 13-cis retinal forms of BR and ASR, akin to those reported for rhodopsin, current results challenge many of the mechanisms held responsible for the ballistic photochemical dynamics observed in visual pigment.

Electron transport (ETp) across bacteriorhodopsin (bR), a natural proton pump protein, in the solid state (dry) monolayer configuration, was studied as a function of temperature. Transport changes from thermally activated at T > 200 K to temperature independent at

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

A VIS pump/hyperspectral NIR probe study of all-trans-retinal protonated Schiff base (RPSB) in ethanol is presented. Upon irradiation, a short-lived absorption band covers the recorded range of λ = 1-2 μm. It decays to reveal the tail of S ₁ emission at λ 0. The existence of this hitherto unrecorded excited-state absorption deep in the NIR will require a revision of current models for RPSB electronic structure. The phenomenological similarity of these observations with ultrafast NIR studies of carotenoids raises the question of whether three, and not two, electronic states participate in RPSB photochemistry as well. The relevance of these observations to retinal protein photochemistry is discussed.

Studying solid-state electronic conductance of biological molecules requires interfacing the biomolecules with electronic conductors without altering the molecules. To this end, we developed and present here a simple, solution-based approach of conjugating Bacteriorhodopsin (bR)-containing membranes with metallic clusters. Our approach is based on selective electroless deposition of Pt nanoparticles on suspended membrane fragments through chemical interaction of the Pt precursor with the proteins residues. Optical absorption measurements show that the membranes retain their photoactivity after this procedure. The result of the Pt deposition is best shown by conductive probe atomic force microscopy mapping of electronic current transport across such soft biological layers, which allows reproducible microscopic electrical characterization of the electronic conductance of the resulting junctions. The maps show that chemical contact between the protein and the deposited electrode yields better electronic coupling than a physical contact, demonstrating that also with biomolecules, the type and method of deposition of the electrical contact are critical to the behavior of the resulting junctions.

Proteorhodopsin (PR), a retinal protein of marine proteobacteria, is a light-driven proton pump. Light excitation of PR initiates a photocycle that triggers the translocation of a proton from the cytoplasmic to the extracellular side. Asp97 is located near the retinal-protonated Schiff base and serves as the proton acceptor during the photocycle. The pK_a of Asp97 is unusually high (∼7.0), especially in comparison with that of its bR equivalent residue Asp85 (∼2.6). We have studied possible anions binding to PR (produced from gene vector eBAC31A08 and expressed in Escherichia coli) and their effect on its absorption maxima, Asp97 pK_a, and the photocycle. We found that chloride, sulfate, trifluoroacetate, trichloroacetate, and tribromoacetate anions bind to PR and regulate Asp97 pK_a. Asp97 has a pK_a of ∼7.8 in water, but the value decreases to ∼7.0 in the presence of sulfate and chloride anions. Halogeno-acetate anions elevated Asp97 pK_a and compete with chloride anions. The most significant effect was detected with tribromoacetate anions that increase the Asp97 pK_a to a value of >9.5. The possibility that PR has at least two binding sites for these anions is discussed. In addition, we have demonstrated that these anions bind to PR also at high pH (above Asp97 pK_a) because they affect the rate of growth and thermal decay of the M intermediate in the photocycle of PR.

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.

Femtosecond pump, NIR dump experiments demonstrate that contrary to previous reports, nonexponential internal conversion in Natronomonas pharaonis Halorhodopsin doesn't reflect bifurcation in the fluorescent state to short lived reactive, and slowly decaying non reactive populations.

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

Essential for the biological function of the light-driven proton pump, bacteriorhodopsin (BR), and the light sensor, sensory rhodopsin II (SRII), is the coupling of the activated retinal chromophore to the hosting protein moiety. In order to explore the dynamics of this process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled BR and SRII samples. These include SRII in D₂O buffer, BR in H₂¹⁸O medium, SRII with ¹⁵N-labeled protein, and BR with ¹³C₁₄¹³C₁₅-labeled retinal chromophore. Via observed shifts of infrared difference bands after photoexcitation and their kinetics we provide evidence for nonchromophore bands in the amide I and the amide II region of BR and SRII. A band around 1550 cm^-1 is very likely due to an amide II vibration. In the amide I region, contributions of modes involving exchangeable protons and modes not involving exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to bending vibrations of protein-bound water molecules. The observed protein bands appear in the amide I region within the system response of ca. 0.3 ps and in the amide II region within 3 ps, and decay partially in both regions on a slower time scale of 9-18 ps. Similar observations have been presented earlier for BR5.12, containing a nonisomerizable chromophore (R. Gross et al. J. Phys. Chem. B 2009, 113, 7851-7860). Thus, the results suggest a common mechanism for ultrafast protein response in the artificial and the native system besides isomerization, which could be induced by initial chromophore polarization.

Xanthorhodopsin (xR) is a recently discovered retinal protein that contains, in addition to the retinal chromophore, a carotenoid (salinixanthin) absorbing at 456, 486, and 520 nm, which functions as a light-harvesting antenna. We have studied the interactions between the two chromophores by monitoring the absorbance and circular dichroism (CD) spectroscopies of artificial pigments derived from synthetic retinal analogues characterized by shifted absorption maxima. In addition, we have followed the binding process of the synthetic chromophores to the apomembrane of xR. We have revealed that the CD spectrum of xR originated mainly from the carotenoid chromophore without a significant contribution of the retinal chromophore. Because the binding process rate of these analogues is slower compared to all-trans retinal, it was possible to detect and analyze the major alterations in the CD spectrum. It was revealed that the main changes occur as a result of binding site occupation by the retinal chromophore and not because of the formation of the retinal-protein covalent bond.

Bacteriorhodopsin, reconstituted with a sterically "locked" retinal chromophore, BR5.12, has frequently been used to elucidate elementary photoinduced processes in the native pigment bacteriorhodopsin. In this work, the vibrational response of BR5.12 to photoexcitation is investigated by means of femtosecond time-resolved mid-infrared and UV-vis spectroscopy. The electronically excited state of BR5.12 decays with a time constant of 18 ps. Neither in the UV-vis nor in the mid-IR spectral region are indications found for chromophore photoproducts, besides the full recovery of the electronic ground state. However, vibrational bands are observed at around 1660 and 1550 cm^-1 in the protein amide I and amide II band regions, respectively. They are formed within a few picoseconds or even instantaneously. Thus, they appear faster than the Si decay and persist for at least 130 ps, i.e., for much longer than the Si lifetime. These findings strongly suggest that the observed bands must be assigned to protein vibrations and that they are not caused by a photoinduced temperature rise. Thus, for the first time, ultrafast protein vibrational changes are detected in BR5.12, that are not associated with isomerization. Possibly they can be related to the enhanced chemical reactivity of photoactivated BR5.12 reported in the literature. In wild-type bacteriorhodopsin, bands with very similar spectral and kinetic characteristics are observed, suggesting that they might originate from a similar mechanism which is not isomerization. A plausible mechanism is a polarization induced protein conformational change, as discussed in the literature.

The second extracellular loop (EL2) of rhodopsin forms a cap over the binding site of its photoreactive 11-cis retinylidene chromophore. A crucial question has been whether EL2 forms a reversible gate that opens upon activation or acts as a rigid barrier. Distance measurements using solid-state ¹³C NMR spectroscopy between the retinal chromophore and the β4 strand of EL2 show that the loop is displaced from the retinal binding site upon activation, and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular ends of transmembrane helices H4, H5 and H6. NMR measurements further reveal that structural changes in EL2 are coupled to the motion of helix H5 and breaking of the ionic lock that regulates activation. These results provide a comprehensive view of how retinal isomerization triggers helix motion and activation in this prototypical G protein-coupled receptor.

Interfacing functional proteins with solid supports for device applications is a promising route to possible applications in bio-electronics, -sensors, and -optics. Various possible applications of bacteriorhodopsin (bR) have been explored and reviewed since the discovery of bR. This tutorial review discusses bR as a medium for biomolecular optoelectronics, emphasizing ways in which it can be interfaced, especially as a thin film, solid-state current-carrying electronic element.

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.

Photochemistry of protonated all-trans retinal Schiff-base (RPSB), the active chromophore in bacteriorhodopsin (BR) and sensory rhodopsins has been investigated with femtosecond multichannel pump probe spectroscopy at two excitation wavelengths. In a recent study of an RPSB analogue which mimics the opsin shift in BR, significant excitation wavelength dependence of the transient spectra was observed and assigned to structural inhomogeneity in the ground state. Our aim is to determine if similar inhomogeneity is manifest also in the native RPSB in solution which is the archtypical model for appreciating the apoproteins effect on retinal protein photochemistry. Significant differences in transient spectra collected after 390 and 480 nm excitation are observed and are likewise assigned to ground state structural inhomogeneity. For both excitation wavelengths the stimulated emission band extends well beyond 900 nm, much deeper than previously reported in the near IR. The shallowness of this feature and a newly revealed dip in its intensity near 760 nm are attributed to an overlapping excited state absorption, as reported for BR. This assignment identifies the documented RPSB excited state absorption band which peaks at 500 nm as the counterpart of the 460 nm absorption feature reported for the reactive excited state of BR coined I₄₆₀. Implications of this assignment, and possible mechanisms for inhomogeneous broadening of the electronic absorption spectrum of RPSB in solution are discussed.

A reliable and reproducible method for preparing bacteriorhodopsin (bR)-containing metal-biomolecule-monolayer-metal planar junctions via vesicle fusion tactics and soft deposition of Au top electrodes is reported. Optimum monolayer and junction preparations, including contact effects, are discussed. The electron-transport characteristics of bR-containing membranes are studied systematically by incorporating native bR or artificial bR pigments derived from synthetic retinal analogues, into single solid-supported lipid bilayers. Current-voltage (I-V) measurements at ambient conditions show that a single layer of such bR-containing artificial lipid bilayers pass current in solid electrode/bilayer/solid electrode structures. The current is passed only if retinal or its analogue is present in the protein. Furthermore, the preparations show photoconductivity as long as the retinal can isomerize following light absorption. Optical characterization suggests that the junction photocurrents might be associated with a photochemically induced M-like intermediate of bR. I-V measurements along with theoretical estimates reveal that electron transfer through the protein is over four orders of magnitude more efficient than what would be estimated for direct tunneling through 5 nm of water-free peptides. Our results furthermore suggest that the light-driven proton-pumping activity of the sandwiched solid-state bR monolayer contributes negligibly to the steady-state light currents that are observed, and that the orientation of bR does not significantly affect the observed I-V characteristics.

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.

Coupling of noble-metal nanoparticles (NPs) into electronics might lead to interesting new avenues in nanoelectronics. It was found that currents through metal//organic (or inorganic) insulator/Au NP monolayer//metal planar junctions (2) are orders of magnitude higher than what is obtained without the NP monolayer (1). (Graph Presented).

Studying electron transport (ET) through proteins is hampered by achieving reproducible experimental configurations, particularly electronic contacts to the proteins. The transmembrane protein bacteriorhodopsin (bR), a natural light-activated proton pump in purple membranes of Halobacterium salinarum, is well studied for biomolecular electronics because of its sturdiness over a wide range of conditions. To date, related studies of dry bR systems focused on photovoltage generation and photoconduction with multilayers, rather than on the ET ability of bR, which is understandable because ET across 5-nm-thick, apparently insulating membranes is not obvious. Here we show that electronic current passes through bR-containing artificial lipid bilayers in solid "electrode-bilayer-electrode" structures and that the current through the protein is more than four orders of magnitude higher than would be estimated for direct tunneling through 5-nm, water-free peptides. We find that ET occurs only if retinal or a close analogue is present in the protein. As long as the retinal can isomerize after light absorption, there is a photo-ET effect. The contribution of light-driven proton pumping to the steady-state photocurrents is negligible. Possible implications in view of the suggested early evolutionary origin of halobacteria are noted.

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

Using Fourier transform infrared (FTIR) difference spectroscopy, we have studied the impact of sites and extent of methylation of the retinal polyene with respect to position and thermodynamic parameters of the conformational equilibrium between the Meta I and Meta II photoproducts of rhodopsin. Deletion of methyl groups to form 9-demethyl and 13-demethyl analogues, as well as addition of a methyl group at C10 or C12, shifted the Meta I/Meta II equilibrium toward Meta I, such that the retinal analogues behaved like partial agonists. This equilibrium shift resulted from an apparent reduction of the entropy gain of the transition of up to 65%, which was only partially offset by a concomitant reduction of the enthalpy increase. The analogues produced Meta II photoproducts with relatively small alterations, while their Meta I states were significantly altered, which accounted for the aberrant transitions to Meta II. Addition of a methyl group at C14 influenced the thermodynamic parameters but had little impact on the position of the Meta I/Meta II equilibrium. Neutralization of the residue 134 in the E134Q opsin mutant increased the Meta II content of the 13-demethyl analogue, but not of the 9-demethyl analogue, indicating a severe impairment of the allosteric coupling between the conserved cytoplasmic ERY motif involved in proton uptake and the Schiff base/Glu 113 microdomain in the 9-demethyl analogue. The 9-methyl group appears therefore essential for the correct positioning of retinal to link protonation of the cytoplasmic motif with protonation of Glu 113 during receptor activation.

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

Halorhodopsin from Natronomonas pharaonis is a light-driven chloride pump which transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. It was shown that the chloride anion bound in the vicinity of retinal PSB can be replaced by several inorganic anions, including azide which converts the chloride pump into a proton pump and induces formation of an M-like intermediate detected in the bR photocycle but not in native halorhodopsin. Here we have studied the possibility of replacing the chloride anion with organic anions and have followed the photocycle under several conditions. It is revealed that the chloride can be replaced with a formate anion but not with larger organic anions such as acetate. Flash photolysis experiments detected in the formate pigment an M-like intermediate characterized by a lifetime much longer than that of the O intermediate. The lifetime of the M-like intermediate depends on the pH, and its decay is significantly accelerated at low pH. The decay rate exhibited a titration-like curve, suggesting that the protonation of a protein residue controls the rate of M decay. Similar behavior was detected in N. pharaonis pigments in which the chloride anion was replaced with NU₂^- and OCN^- anions. It is suggested that the formation of the M-like intermediate indicates branching pathways from the L intermediate or basic heterogeneity in the original pigment.

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.

Proteorhodopsin, a retinal protein of marine proteobacteria similar to bacteriorhodopsin of the archaea, is a light-driven proton pump. Absorption of a light quantum initiates a reaction cycle (turnover time of ca. 50 ms), which includes photoisomerization of the retinal from the all-trans to the 13-cis form and transient deprotonation of the retinal Schiff base, followed by recovery of the initial state. We report here that in addition to this fast cyclic conversion, illumination at high pH results in accumulation of a long-lived photoproduct absorbing at 362 nm. This photoconversion is much more efficient in the D227N mutant in which the anionic Asp227, which together with Asp97 constitutes the Schiff base counterion, is replaced with a neutral residue. Upon illumination at pH 8.5, most of the D227N pigment is converted to the 362 nm species, with a quantum efficiency of ca. 0.2. The pK(a) for this transition in the wild type is 9.6, but decreased to 7.5 after mutation of Asp227. The short wavelength of the absorption maximum of the photoproduct indicates that it has a deprotonated Schiff base. In the dark, this photoproduct is converted back to the initial pigment with a time constant of 30 min (in D227N, at pH 8.5), but it can be reconverted more rapidly by illumination with near-UV light. Experiments with "locked" retinal analogues which selectively exclude rotation around either the C9=C10, C11=C12, or C13=C14 bond show that formation of the 362 nm species involves isomerization around the C13=C14 bond. In agreement with this, retinal extraction indicates that the 362 nm photoproduct is 13-cis whereas the initial state is predominantly all-trans. A rapid shift of the pH from 8.5 to 4 greatly accelerates thermal reconversion of the 362 nm species to the initial pigment, suggesting that its recovery involving the thermal isomerization of the chromophore is controlled by ionizable residues, primarily the Schiff base and Asp97. The transformation to the long-li

The bacteriorhodopsin (bR) monolayers were prepared by a self-assembly procedure on a solid surface. The orientation of monolayers on an Al substrate was determined using a Kelvin probe. The good orientation of purple membrane (PM) patches to the electrostatic asymmetry between the two sides of the membrane protein was described. Such monolayers, made from WT bR, exhibited a photoelectric response that was quite different from that of the bR multilayer and the bR suspension. It was observed that upon green-light illumination, the characteristic absorption of bR, with a maximum at ∼560 nm disappeared and a maximum at ∼410 nm appeared, indicating the formation of the M intermediate.

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.

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

Binding of arrestin to light-activated rhodopsin involves recognition of the phosphorylated C-terminus and several residues on the cytoplasmic surface of the receptor. These sites are in close proximity in dark, unphosphorylated rhodopsin. To address the position and mobility of the phosphorylated C-terminus in the active and inactive receptor, we combined high-resolution solution and solid state NMR spectroscopy of the intact mammalian photoreceptor rhodopsin in detergent micelles as a function of temperature. The ³¹P NMR resonance of rhodopsin phosphorylated by rhodopsin kinase at the C-terminal tail was observable with single pulse excitation using magic angle spinning until the sample temperature reached -40 °C. Below this temperature, the ³¹P resonance broadened and was only observable using cross polarization. These results indicate that the phosphorylated C-terminus is highly mobile above -40 °C and immobilized at lower temperature. To probe the relative position of the immobilized phosphorylated C-terminus with respect to the cytoplasmic domain of rhodopsin, ¹⁹F labels were introduced at positions 140 and 316 by the reaction of rhodopsin with 2,2,2-trifluoroethanethiol (TET). Solid state rotational-echo double-resonance (REDOR) NMR was used to probe the internuclear distance between the ¹⁹F and the ³¹P-labels. The REDOR technique allows ¹⁹F⋯³¹P distances to be measured out to ∼12 Å with high resolution, but no significant dephasing was observed in the REDOR experiment in the dark or upon light activation. This result indicates that the distances between the phosphorylated sites on the C-terminus and the ¹⁹F sites on helix 8 (Cys 316) and in the second cytoplasmic loop (Cys140) are greater than 12 Å in phosphorylated rhodopsin.

The special trimeric structure of bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarum, and especially, the still controversial question as to whether the three protein components are structurally and functionally identical, have been subject to considerable work. In the present work, the problem is approached by studying the reconstitution reaction of the bR apo-protein with all-trans retinal, paying special attention to the effects of the apo-protein/retinal (P:R) ratio. The basic observation is that at high P:R values, the reconstitution reaction proceeds via two distinct, fast and slow, pathways associated with two different pre-pigment precursors absorbing at 430 nm (P430) and 400 nm (P₄₀₀), respectively. These two reactions, exhibiting 2:1 (P₄₃₀/P₄₀₀) amplitude ratios, are markedly affected by the P:R value. The principal feature is the acceleration of the P₄₀₀ → bR transition at low P:R ratios. The data are interpreted in terms of a scheme in which the added retinal first occupies two protein retinal traps, R₁ and R₂, from which it is transferred to two spectroscopically distinct binding sites corresponding to the two pre-pigments, P₄₃₀ and P₄₀₀, respectively. Two noncovalently bound retinal molecules occupy two P₄₃₀ sites of the bR trimer, while one (P₄₀₀) occupies the third. Binding is completed by generating the retinal-protein covalent bond. Analogous experiments were also carried out with an aromatic bR chromophore and with the D85N bR mutant. The accumulated data clearly point out the heterogeneity of the binding reaction intermediates, in which two are clearly distinct from the third. However, CD spectroscopy strongly suggests that even the two P430 sites are not structurally identical. The heterogeneity of the P intermediates in the binding reaction can be accounted for, either by being induced by cooperativity or by an intrinsic heterogeneity that is already present in the apoprotein. The question as to whether the final reconstituted pigment, as well as native bR, are nonhomogeneous should be the subject of future studies.

Following light absorption, the retinal chromophore of bacteriorhodospin experiences very large dipolar changes in the vertically excited state. This light-induced dipole is at least 50% larger than that of the retinal chromophore in films or in solution. We have studied the origin of the protein effect by applying second harmonic generation measurements of artificial bacteriorhodopsin pigments derived from synthetic retinal analogues, characterized by a modified polyene chain length and ring-chain conformation. The studies demonstrated a significant influence of a protein domain in the vicinity of the retinal β-ionone. We suggest that tryptophane residues (specifically Trp 138 and 189) enhance the light-induced dipole in bacteriorhodopsin. The data also point to another protein domain (Trp 182) influencing the retinal chromophore. The effect of these tryptophane residues appears not only to stabilize the light-induced charge redistribution but also to enable the migration in the excited state of the positive charge into a region of the protein with a relatively low dielectric constant.

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

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.

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

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.

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.

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.

In rhodopsin, the retinal chromophore is covalently bound to the apoprotein by a protonated Schiff base, which is stabilized by the negatively charged counterion Glu¹¹³, conferring upon it a pK_a of presumably > 16. Upon photoexcitation and conformational relaxation of the initial photoproducts, the Schiff base proton neutralizes the counterion, a step that is considered a prerequisite for formation of the active state of the receptor, metarhodopsin II (MII). We show that the pK_a of the Schiff base drops below 2.5 in MII. In the presence of solute anions, however, it may be increased considerably, thereby leading to the formation of a MII photoproduct with a protonated Schiff base (PSB) absorbing at 480 nm. This PSB is not stabilized by Glu¹¹³, which is shown to be neutral, but by stoichiometric binding of an anion near the Schiff base. Protonation of the Schiff base in MII changes neither coupling to G protein, as assessed by binding to a transducin-derived peptide, nor the conformation of the protein, as judged by FTIR and UV spectroscopy. A PSB and an active state conformation are therefore compatible, as suggested previously by mutants of rhodopsin. The anion specificity of the stabilization of the PSB follows the series thiocyanate > iodide > nitrate > bromide > chloride > sulfate in order of increasing efficiency. This specificity correlates inversely with the strength of hydration of the respective anion species in solution and seems therefore to be determined mainly by its partitioning into the considerably less polar protein interior.

In this study, the ultrafast pump-probe spectroscopy of the all-trans protonated Schiff base of retinal (trans-PSB) in solution, is compared to that of two retinal analogues, trans-PSB5.12 and 13-cis-PSB5.13, in which C₁₃=C₁₄ torsional motion is inhibited by a rigid five-membered ring structure. The objective is to obtain measures of internal conversion (IC) dynamics in these polyenes. Contrasting the results with those obtained for the same pigments when attached to their opsin protein, serve to appreciate the protein role in catalyzing energy transduction in bacteriorhodopsin. Several major features appear to be common to all three PSBs: (i) A 50-100 fs process due to a primary relaxation out of the Franck-Condon (FC) region, (ii) A subsequent biexponential decay (t₁ = 1-2 ps and t₂ = 4-7 ps) of the fluorescent state (FS) assumed to be due to IC, and (iii) Spectral modulations in the FS emission. The three are only marginally effected by locking of the C₁₃=C₁₄ bond. With respect to features (i) and (iii) the PSB model compounds behave analogously to the related retinal protein bacteriorhodopsin (bR). However, this does not apply to the FS decay. While in bR, the IC takes place with a 0.5 ps decay time, locking of the C₁₃=C₁₄ bond in bR markedly increases the FS lifetime to ∼ 15 ps. These observations demonstrate the crucial role played by the protein in directing the isomerization action to the active double bond and enhancing the rate of IC. They also prove that these coordinates are not exclusive pathways of IC in the isolated PSB of retinal. The mechanism of ground-state repopulation in the PSBs is discussed in light of these results.

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 formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.

The vibrational spectrum of an intermediate, T5.12, in the photoreaction of an artificial bacterio-rhodopsin (BR) pigment containing a five-membered carbon ring spanning the C₁₂-C₁₃=C₁₄ bonds (BR5.12) is measured by picosecond time-resolved coherent anti-Stokes Raman spectroscopy (PTR/CARS). Observed initially by picosecond transient absorption (PTA) measurements, T5.12 is the only intermediate in the BR5.12 photoreaction (i.e., T5.12 decays only to BR5.12). BR5.12 does not have a photocycle analogous to that in native BR, presumably because the five-membered ring blocks the reaction coordinate leading to C₁₃=C₁₄ bond isomerization. Since T5.12 may therefore represent the molecular events (reaction coordinates) that precede C₁₃=C₁₄ bond isomerization, its vibrational spectrum may aid in elucidating the primary reaction coordinate(s) in the BR photocycle. Although T5.12 is identified via a red-shifted absorption (660 nm maximum,

Stimulated emission from excited bacteriorhodopsin is compared with that from a isomerize. Ultrafast Stokes shifting of emission, and coherent vibrations in the excited state are observed for the first time. Biological relevance of coherent motions is discussed.

We report the results of experiments on the application of electric fields across thin, dry films of a bacteriorhodopsin (BR) analog pigment in which the retinal chromophore has been replaced with 13-demethyl-11,14-epoxyretinal. As previously observed in other BR variants with low Schiff-base pK values, this pigment exhibits protonation and deprotonation of the Schiff base under an applied electric field, depending on the initial Schiffbase protonation state or effective pH. At low effective pH, a fast (

An outstanding problem relating to the structure and function of bacteriorhodopsin (bR), which is the only protein in the purple membrane of the photosynthetic microorganism Halobacterium salinarium, is the relation between the titration of Asp-85 and the binding/unbinding of metal cations. An extensively accepted working hypothesis has been that the two titrations are coupled, namely, protonation of Asp-85 (located in the vicinity of the retinal chromophore) and cation unbinding occur concurrently. We have carried out a series of experiments in which the purpleblue equilibrium and the binding of Mn²⁺ ions (monitored by electron spin resonance) were followed as a function of pH for several (1-4) R=[Mn²⁺]/[bR] molar ratios. Data were obtained for native bR, bR mutants, artificial bR and chemically modified bR. We find that in the native pigment the two titrations are separated by more than a pK(a) unit [ΔpK(a)=pK(a)(P/B)-pK(a)(Mn²⁺)=(4.2-2.8)=1.4]. In the non-native systems, ΔpK(a) values as high as 5 units, as well as negative ΔpK(a)s, are observed. We conclude that the pH titration of cation binding residues in bR is not directly related to the titration of Asp-85. This conclusion is relevant to the nature of the high affinity cation sites in bR and to their role in the photosynthetic function of the pigment. Copyright (C) 1999 Federation of European Biochemical Societies.

Replacing the chromophore of bacteriorhodopsin with chemically modified retinal analogs which cannot isomerize, allows to measure directly side effects from the laser pulse used for sample excitation in step-scan FT-IR measurements. Comparison with static temperature difference spectra and temperature-jump experiments shows that the observed effects can mainly be attributed to heating of the sample by the laser pulse. The size of resulting spectral changes is compared to difference bands of the photoreaction.

Nanosecond laser photolysis measurements were conducted on digitonin extracts of artificial pigments prepared from the cone-type visual pigment, P521, of the Tokay gecko (Gekko gekko) retina. Artificial pigments were prepared by regeneration of bleached gecko photoreceptor membranes with 9- cis-retinal, 9-cis-14-methylretinal, or 9-cis-α-retinal. Absorbance difference spectra were recorded at a sequence of time delays from 30 ns to 60 μs following excitation with a pulse of 477-nm actinic light. Global analysis showed the kinetic data for all three artificial gecko pigments to be best fit by two-exponential processes. These two-exponential decays correspond to similar decays observed after photolysis of P521 itself, with the first process being decay of the equilibrated P521 Batho mutually implies P521 BSI mixture to P521 Lumi and the second process being the decay of P521 Lumi to P521 Meta I. In spite of its large blue shift relative to P521, iso- P521 displays a normal chloride depletion induced blue shift. Iso-P521's early intermediates up to Lumi were also blue-shifted, with the P521 Batho mutually implies P521 BSI equilibrated mixture being 15 nm blue-shifted and P521 Lumi being 8 nm blue-shifted relative to the intermediates formed after P521 photolysis. The blue shift associated with the iso-pigment is reduced or disappears entirely by P521 Meta I. Similar blue shifts were observed for the early intermediates observed after photolysis of bovine isorhodopsin, with the Lumi intermediate blue-shifted 5 nm compared to the Lumi intermediate formed after photolysis of bovine rhodopsin. These shifts indicate that a difference exists between the binding sites of 9- and 11-cis pigments which persists for microseconds at 20 °C.

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.

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.

The thermal re-isomerization of retinal from the 13-cis to the all- trans state is a key step in the final stages of the photocycle of the light- driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu- 93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 → Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.

Upon light adaptation by continuous (or pulsed) illumination, the artificial bacteriorhodopsin (bR) pigments, I and II, derived from synthetic 14F retinal and a short polyenal, respectively produce a long-lived red-shifted species denoted O-1. An analogous phenomenon was observed by Sonar, S., et al. [(1993) Biochemistry 32, 2263-2271], in the case of the Y185F mutant (pigment III). The nature of these O-1 species was investigated by studying a series of effects, primarily their red Light photoreversibility, the associated proton uptake and release processes, and the effects of pH on their relative amounts, which are interpreted in terms of pH-dependent acid-base equilibria. Experiments were also carried out with pigments I and II derived from the mutants D96A, E204Q, R82Q, and D85N. The O-1 species of pigments I and II. (and possibly also that of pigment III) are identified as an unusually long-lived (all-trans) intermediate of the photocycle of their 13-cis isomer. It is concluded that in O-1, Asp-85 is protonated, a process associated with proton uptake from the extracellular side. Subsequent proton release (to the same side of the membrane) occurs from Glu-204 (or from a group closely interacting with it) prior to the decay of O-1. At high pH (>9), O-1 reversibly converts to a purple form, due to deprotonation of Asp-85, while at still higher pH (>11), a blue-shifted species characterized by a deprotonated Schiff base is generated. These transitions constitute the first demonstration of the titration of a photocycle intermediate of a retinal protein. The respective pK(a) values are determined and discussed in relation to those pertaining to the unphotolyzed (dark-adapted) pigments. It appears that the pK, values are controlled by a hydrogen bond network involving water molecules, which binds the protonated Schiff base with Asp-85 and Glu-204. The disruption of this network in pigments I-III may also be responsible for the long lifetime of the O-1 species, due

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

Three experimental observations indicate that the pK(a) of the purple- to-blue transition (the pK(a) of Asp-85) is higher for all-trans-bR¹ than for 13-cis-bR. First, light adaptation of bacteriorhodopsin (bR) at pHs near the pK(a) of Asp-85 causes an increase in the fraction of the blue membrane present. This transformation is reversible in the dark. Second, the pK(a) of the purple-to-blue transition in the dark is lower than that in the light- adapted bR (pK(a)/(DA) = 3.5, pK(a)/(LA) = 3.8 in 10 mM K₂SO₄). Third, the equilibrium fractions of 13-cis and all-trans isomers are pH dependent; the fraction of all-trans-bR increases upon formation of the blue membrane. Based on the conclusion that thermal all-trans 4⇆ 13-cis isomerization occurs in the blue membrane rather than in the purple, we have developed a simple model that accounts for all three observations. From the fit of experimental data we estimate that the pK(a) of Asp-85 in 13-cis-bR is 0.5 ± 0.1 pK(a) unit less than the pK(a) of all-trans-bR. Thus, in 10 mM K₂SO₄, pK(a)/(c) = 3.3, whereas pK(a)/(t) = 3.8.

This paper demonstrates that an atomic force microscope can be used to directly monitor rapid membrane protein dynamics. For this demonstration the membrane-bound proton pump, bacteriorhodopsin, has been investigated. It has been unequivocally shown that the light-induced dynamic alterations that have been observed do not arise from external artifacts such as heating of the sample by the incident light, but that these changes can be directly linked to the light-induced protein conformational alterations in this membrane. In essence, it has been shown that the light energy absorbed by bacteriorhodopsin is converted not only to chemical energy but also to mechanical energy. In summary a new ultrasensitive tool is described for monitoring the molecular dynamics of materials with wide applicability to fundamental and applied science.

Ultrafast excitation of native and artificial bacteriorhodopsin leads to a revision of the currently accepted photophysical models based on a ≤ 100 fs C₁₃=C₁₄ isomerization of the retinal chromophore.

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.

Tyrosine 57 is one of the residues present in the retinal binding pocket and is conserved in all the halophilic retinal proteins. We have studied mutants of bacteriorhodopsin, expressed in Halobacterium salinarium, in which tyrosine 57 is replaced by an asparagine (Y57N) or phenylalanine (Y57F). In Y57N the photocycle proceeds only up to the L intermediate; no M is formed at neutral pH. The lifetime of L intermediate is extremely long, ca. 500 ms. Proton release is severely affected in both the mutants which suggests that Y57 is associated with the proton release pathway. By comparing the pH-induced absorption changes in the UV in Y57N and Y57F with those in the wild-type (WT), we determined that the pK_a of Y57 is 10.2. In Y57F, which shows M formation, the rate constant of the L → M transition is pH dependent (pk_a 8.7) suggesting that Y57 is probably not the residue that normally controls the transition into the alkaline photocycle. Y57 is either part of the counterion complex or in close proximity to D85 since its mutation influences the pK_a of Asp85. In Y57F the pK_a of D85 is ~ 4.9 (compared to & 2.9 in the WT). The Y57N mutant shows two pK_a's in the purple to blue transition, ~3.8 and

Kinetic spectra of early photolysis intermediates were monitored after nanosecond laser photolysis of a series of artificial visual pigments containing retinal analogs with bulky substituents along the polyene chain. Time-resolved absorbance changes over the spectral range 400-700 nm were recorded at discrete times from 20 ns to 5 μs following room temperature excitation with a pulse of 477 nm light. Photolysis of bovine rhodopsin regenerated with 9-ethyl-9-cis-retinal, 19,11-ethano-11-cis-retinal, or 13-ethyl-9-cis-retinal produced intermediates similar to those seen after rhodopsin photolysis, i.e. bathorhodopsin (Batho) ⇆ blue-shifted intermediate (BSI) → lumirhodopsin (Lumi). In contrast to previously studied artificial pigments and rhodopsin itself, for these chromophores with bulky substituents, the equilibrium between Batho and BSI is back-shifted. The stability of BSI relative to Batho is most affected in the 13-ethyl pigment, which had an equilibrium constant of 0.4, approximately one-third of the value observed for rhodopsin. As the bulky substituent moves toward the G9 end of the chromophore, K(eq) moves toward the rhodopsin value, with the result for the locked 9-trans-rhodopsin pigment being intermediate between those of the 9-ethyl and 13-ethyl pigments. The presence of bulky substituents also slows the microscopic rate of Batho decay. This effect is largest for the 9-ethyl pigment whose Batho decay is slowed by a factor of 5. Freedom of movement of the 9-methyl (restricted in the 9-ethyl case) is proposed to control the rate of Batho decay in a mechanism involving passage of the chromophore's C8-hydrogen by the 5-methyl group of its β-ionone ring to form BSI. For all these pigments, the decay of BSI is substantially slower than for previous pigments, indicating that steric hindrance along the polyene chain interferes with the protein change triggered by BSI formation and suggesting that the protein change may involve side chains adjacent to this region of the chromophore.

The availability of the structure of bacteriorhodopsin from electron microscopy studies has opened up the possibility of exploring the proton pump mechanism of this protein by means of molecular dynamics simulations. In this review we summarize earlier theoretical investigations of the photocycle of bacteriorhodopsin including relevant quantum chemistry studies of retinal, structure refinement, molecular dynamics simulations, and evaluation of pK_a values. We then review a series of recent modeling efforts which refined the structure of bacteriorhodopsin adding internal water, and which studied the nature of the J intermediate and the likely geometry of the K₅₉₀ and L₅₅₀ intermediates (strongly distorted 13cis) as well as the sequence of retinal geometry and protein conformational transitions which are conventionally summarized as the M₄₁₂ intermediate. We also review simulations of the photocycle of lightadapted bacteriorhodopsin at T=77 K and of the photocycle of darkadapted bacteriorhodopsin, both cycles differing from the conventional photocycle through a nonfunctional (pure 13cis) retinal geometry of the corresponding K₅₉₀ and L₅₅₀ states. The simulations demonstrate a potentially critical role of water and of minute reorientations of retinal's Schiff base nitrogen in controlling proton pumping in bR₅₆₈; the simulations also indicate the existence of heterogeneous photocycles. The results exemplify the important role of molecular dynamics simulations in extending investigations on bacteriorhodopsin to a level of detail which is presently beyond experimental resolution, but which needs to be known to resolve the pump mechanism of bacteriorhodopsin. Finally, we outline the major existing challenges in the field of bacteriorhodopsin modeling.

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.

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.

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.

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.

Using twodimensional NMR spectroscopy, the spatial location of various carboxylate anions relative to the polyene chain of the protonated Schiff base of alltransretinal was determined. The observed intermolecular NOE crosspeaks between a proton on the counter ion and a proton near the nitrogen atom indicate the existence of ionpair formation between the protonated retinal Schiff base and various counter ions in chloroform. The results suggest that the most likely site of the carboxylate group of the counter ion is in the immediate vicinity of the positively charged nitrogen atom of the retinal Schiff base.

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.

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.

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

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.