Effect of metal ion exchange on the photocurrent response from bacteriorhodopsin on tin oxide electrodes

The transient photocurrent response from bacteriorhodopsin (bR) on tin oxide electrodes was strongly influenced by metal ions bound to bR molecules. The photocurrent polarity reversal pH, which corresponded to the pH value for the reversal of the proton release/uptake sequence in the bR photocycle, of cation-substituted purple membrane (PM) was shifted to lower pH with the increase in the cation affinities to carboxyl groups and a close correlation was noted between the two values. This suggests that the metal ion present in the extracellular region of a bR molecule modulates the pK(a) of proton release groups of bR by stabilizing the ionized state of the proton-releasing glutamic acids. The behavior of photocurrents at light-off in alkaline media, reflecting the proton uptake by bR, was unchanged by binding monovalent (Na(+) and K(+)) or divalent cations (Mg(2+) and Ca(2+)), but was drastically changed by binding La(3+) ions. This can be explained by invoking a substantial slowing of the proton uptake process in the presence of La(3+).     

TIME-RESOLVED ULTRAVIOLET ABSORPTION CHANGES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN*

Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time-resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non-chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time-resolved UV-VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob̊ transition.     

细菌视紫红质／聚乙烯醇复合膜的制备及相关功能研究

细菌视紫红质（bR)是一种独特的光敏蛋白，具有光致变色和光驱质子泵功能 。将bR蛋白包埋于聚乙烯醇（PVA）基质中，制备了bR/PVA复合膜。利用紫外－可 见分光光度计和自制的毫秒级动力学光谱仪，检测了样品的吸收光谱和光循环M中 间体在脉冲光激发下随时间的变化；同时，利用凝胶扫描成像仪及相关分析软件考 察了样品成膜后的均匀程度。实验表明：bR/PVA复合膜具有良好的均匀性、透明性 和力学性能，而且bR蛋白保持了原有的生物活性和光学性质，bR与M中间体之间能 达到一种光可控制的双稳态，M中间体的寿命也得到了显著的延长，证实了bR可以 提供一个用于信息存储的模型材料。     

CeO2纳米晶修饰的细菌视紫红质薄膜M态寿命的研究

紫膜中的蛋白质细菌视紫红质(bR)具有独特的光循环和光致变色特性 ,在分子电子学和生物电子技术领域具有广泛的潜在应用价值. 本文采用稀土纳米晶CeO₂对细菌视紫红质 聚乙烯醇(bR-PVA)薄膜进行化学修饰 .研究了纳米晶对bR光循环中的重要中间态M态的寿命的影响. 发现上述稀土纳米晶可延长M态寿命, 且晶粒尺寸越小对M态寿命的影响越大. 纳米晶周围的羟基有助于阻碍席夫碱获得质子, 从而使M态寿命延长.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

LOW TEMPERATURE RESONANCE RAMAN STUDY OF THE L INTERMEDIATE OF BACTERIORHODOPSIN

Abstract— Resonance Raman spectra of photostationary state mixtures at — 80°C of bR568, L, L', and M for light-adapted bacteriorhodopsin were obtained. The approximate Raman spectrum of L + L' was obtained by computer, subtracting the known Raman spectra of bR568 and M from the photostationary state spectra. Several new species not previously observed with the bacteriorhodopsin photocycle have been proposed recently. We compare our results to these studies in order to investigate the photoreaction scheme of bacteriorhodopsin.     

Electric signals of light excited bacteriorhodopsin mutant D96N.

The study of mutant D96N played an important role in understanding proton translocation by light driven bacteriorhodopsin. Our measurement of photoelectric current for single and double flash illumination revealed new details of the photocycle of this mutant. With double flash excitation we found an intermediate absorbing near the wavelength of the ground state of bacteriorhodopsin (bR) but pumping in the opposite direction. This intermediate has the same lifetime as the species described by Zimányi et al. [Proc. Natl. Acad. Sci. USA 96 (1999) 4414-4419] and was assigned to early recovery of a fraction of the ground state after excitation. Because the electric response does not reconcile with that of the ground state, we tentatively assign it to the L intermediate or to an intermediate similar in absorption to bR (bR').     

pH DEPENDENCE OF THE ABSORPTION SPECTRA AND PHOTOCHEMICAL TRANSFORMATIONS OF THE ARCHAERHODOPSINS

Abstract —Two strains of archaebacteria have been found to contain light-driven proton pumping pigments analogous to bacteriorhodopsin (bR) in Halobacterium salinarium . These proteins are called archaerhodopsin-1 (aR-1) and archaerhodopsin-2 (aR-2). Their high degree of sequence identity with bR within the putative proton channel enables us to draw some conclusions about the roles of regions where differences in amino acids exist, and in particular the surface residues, on the structure and function of retinal-based proton pumps. We have characterized the spectral and photochemical properties of these two proteins and compared them to the corresponding properties of bR. While there are some differences in absorbance maxima and kinetics of the photocycle, most of the properties of aR-1 and aR-2 are similar to those of bR. The most striking differences of these proteins with bR are the lack of an alkaline-induced red-shifted absorption species and a dramatic (apparent) decrease in the light-induced transient proton release. In membrane sheet suspensions of aR-1 at 0.15 M KCI, the order of proton release and uptake appears opposite that of bR, in which proton release precedes uptake. The nature of this behavior appears to be due to differences in the amino acid sequence at the surfaces of the proteins. In particular, the residue corresponding to the lysine at position 129 of the extracellular loop region of bR is a histidine in aR-1 and could regulate the efficient release of protons into solution in bR.     

KINETIC MODEL OF BACTERIORHODOPSIN PHOTOCYCLE: PATHWAY FROM M STATE TO bR

A model of the last parts of the bacteriorhodopsin (bR) photocycle is proposed on the basis of experimental data for the kinetic behavior of the 'O' intermediate during a temperature pulse in distilled water suspension. The model includes the previously proposed (but not well characterized) intermediate 'N' between the 'M' and 'O' states of bR. This intermediate exists in fast temperature-dependent quasi-stationary equilibrium with the red-shifted intermediate 'O' and has a maximum of absorption close to the bR spectrum.     

THE EFFECT OF VISCOSITY ON THE PHOTOCYCLE OF BACTERIORHODOPSIN

Abstract— We study the effect of solvent viscosity on the kinetics of the photocycle of bacteriorhodopsin (bR) from Halobacterium halobium. Solvent viscosity is altered by changing the glycerol concentration from 20 to 80% glycerol by volume. The kinetics of the photocycle are observed after flash photolysis at four wavelengths at several temperatures between 240 and 315 K. Assuming a sequential model, bR → K -→ L → M → O → bR, Arrhenius plots of the rate coefficients determine the activation enthalpies and frequency factors for each step. Kinetic data from all solvents are considered together and studied as a function of temperature for fixed solvent viscosities. The early steps of the cycle are insensitive to solvent viscosity, →; the later steps are retarded with increasing viscosity. Activation enthalpies are independent of viscosity; the frequency factors are proportional to η^−K, where the exponent k 0.25 for the transition K → L, 0.0 for L → M, 0.8 for M → O and 0.5 for O → bR.     

Bacteriorhodopsin O-state Photocycle Kinetics: A Surfactant Study

The spectroscopy and dynamics of interaction between the O intermediate of bacteriorhodopsin (bR) and several surfactants (cetrimonium bromide [CTAB], sodium dodecyl sulfate [SDS] and diethylene glycol mono- n -hexyl ether [C6E2]) were investigated using steady-state UV–VIS spectrometry, circular dichroism spectroscopy and time-resolved absorption techniques. The steady-state spectral results show that bR can retain its trimeric state without severe damage in the molar concentration ratio of C6E2/bR ranging up to 4000. Time-resolved observations indicate that the rise and decay rates and transient populations of the O state can be increased in the presence of nonionic surfactant C6E2; however, these studies indicate the opposite phenomenon in the presence of the ionic surfactants CTAB and SDS. The observed 40% enhancement in the transient population of the O intermediate state that results from treatment of C6E2 is proposed to result from an expanding bR structure, which leads to more effective proton pumping efficiency in the photosynthetic system of bR.     

KINETIC ANALYSIS OF TIME-RESOLVED INFRARED DIFFERENCE SPECTRA OF THE L and M INTERMEDIATES OF BACTERIORHODOPSIN*

Abstract– Infrared difference spectra with 4 cm^?1 spectral resolution and 10-u.s temporal resolution, obtained previously with a stroboscopic Fourier-transform difference technique (Braiman et al., 1991, Proc. Natl. Acad. Sci. USA 88, 2388), were analyzed by means of a global exponential fitting procedure based on singular value decomposition. Using a simple linear kinetic model K → L → M for the bacteriorhodopsin (bR) photocycle in the time range10–1000 μ.s at 16.5°C, it was possible to generate bR → L and bR → M difference spectra with signal/noise ratios comparable to those obtainable with low temperature difference spectroscopy. The resulting time-resolved bR → L and bR-→ M difference spectra are both very similar to the corresponding static FTIR difference spectra obtained at 175 K and 250 K, respectively. In the bR → L spectrum, however, there are interesting differences that may indicate a greater degree of deprotonation of Asp-96 when L is formed at physiological temperatures than when it is observed in a low-temperature steady state.     

Photoelectrochemical Verification of Proton-Releasing Groups in Bacteriorhodopsin

Light-induced transient currents of bacteriorhodopsin (bR) and its mutants (D96N, D85N, E204Q and E194Q) were measured at the interface of an electrode and the aqueous solution in an electrochemical cell. The transient positive (cathodic) and negative (anodic) photocurrents generated upon the onsets of continuous illumination and of turning off light, respectively, were investigated at different electrolyte pH. The wild type exhibits both positive and negative responses, with the sign of the response inverted at pH lt 5. In D85N the response is entirely suppressed, while D96N lacks the negative response. Laser pulse excitation (532 nm) of bR and the mutants showed the response rise time of the positive transient to be uniformly about 100 μs, indicating that the response is linked to the L to M transition in the photocycle. According to these results the positive and negative signals originate from the proton release and uptake reactions, respectively. Photoexcitation of the mutants E204Q and E194Q that lack protonatable residues at position 204 and 194, respectively, produced a negative response at neutral pH, which indicates that in these proteins proton uptake precedes proton release. This result is consistent with our observations made earlier with pH indicator dyes and demonstrates that Glu-204 and Glu-194 constitute the terminal proton release complex in the extracellular region of bR.     

TIME-RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR-185→E: ASP-96 REPROTONATES DURING O FORMATION; ASP-85 AND ASP-212 DEPROTONATE DURING O DECAY

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.     

PROTON TRANSLOCATION and CONFORMATIONAL CHANGES DURING THE BACTERIORHODOPSIN PHOTOCYCLE: TIME-RESOLVED STUDIES WITH MEMBRANE-BOUND OPTICAL PROBES and X-RAY DIFFRACTION*

Abstract– For the first time, we monitored time-resolved conformational changes inherent in bacterio-rhodopsin's reaction cycle as well as the H⁺-release events directly at the surface of bacteriorhodopsin (bR) at room temperature. Signals of optical pH-indicators in the aqueous bulk phase were compared with those of probes covalently linked to bR. The kinetics of H⁺-translocation and the correlation of the photocycle with the pumping cycle can only be determined with indicators bound to bR. The proton appears at the extracellular surface during the L₅₅₀ to M₄₁₂ transition. Upon short photo-excitation, diffraction patterns were obtained from both guanidine hydrochloride-treated wild type bR and an Asp96Asn mutant with millisecond time resolution by x-ray synchrotron radiation. The measured time course and location of the structural changes confirm and extend our previous static neutron diffraction study at -180°C. The temporal correlation of photocycle intermediates and proton translocation steps with structural changes in the protein under similar environmental conditions strongly contribute to the understanding of the pumping mechanism.     

Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico- to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light-activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC-module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.     

Bacteriorhodopsin--novel biomolecule for nano devices

The aim of this article is to provide insight on the use of a biological molecule--bacteriorhodopsin (bR) having all the basic properties necessary for the assembly of nanoscale electronic devices. Recent developments made during last decade supported by key references are reviewed in this contribution. Major emphasis on bR-based observations conducted in our laboratory has been elaborated. Important issues concerning structure, widely accepted photocycle of bR has been summarized. The possibility of nano-devices emanating from this biomolecule is briefly presented.     

Characteristic noise features in light transmission across membrane protein undergoing photocycle

We demonstrate a technique based on noise measurements which can be utilized to study dynamical processes in protein assembly. Direct visualization of dynamics in membrane protein system such as bacteriorhodopsin (bR) upon photostimulation are quite challenging. bR represents a model system where the stimulus-triggered structural dynamics and biological functions are directly correlated. Our method utilizes a pump-probe near field microscopy method in the transmission mode and involves analyzing the transmittance fluctuations from a finite size of molecular assembly. Probability density distributions indicating the effects of finite size and statistical correlations appear as a characteristic frequency distribution in the noise spectra of bR whose origin can be traced to photocycle kinetics. Valuable insight into the molecular processes were obtained from the noise studies of bR and its mutant D96N as a function of external parameters such as temperature, humidity or presence of an additional pump source.     

ISOMER COMPOSITION and SPECTRA OF THE DARK and LIGHT ADAPTED FORMS OF ARTIFICIAL BACTERIORHODOPSINS*

Abstract– The isomer composition and spectral properties of 15 artificial bacteriorhodopsin (bR) pigments, based on a series of retinal analogs with polyene residue modified below C₉ are determined for both dark-adapted (DA) and light-adapted (LA) forms. Similarly to native bR, in all cases only two isomers, C₁₃=C₁₄cis (13-cis) and M-trans, are observed. However, the artificial DA pigments have a lower 13-d.s content than native DA bR (? 66%) while the corresponding LA pigments have a much higher 13-cis content (11-69%) than native LA bR (<2%). Thus, in variance with the native pigment, in all of the artificial systems light also induced the reversed all-trans13-cis process. The data are accounted for in terms of specific steric interactions between the polyene and the protein binding site which allow a (C₁₅-anti)(C_ls-syn) isomerization during the photocycle of the artificial pigments, but not in the case of native bR. This accounts for the high proton pumping efficiency of the natural pigment. The nature of a highly red shifted light-adapted form of two of the artificial pigments is investigated and discussed. It is also shown that, in variance with native bR, several artificial pigments exhibit identical absorption spectra for their 13-cis and all-trans isomers. It is concluded that the spectral data for the above species of artificial pigments do not lead to a clear molecular model for the origin of the spectral shift between 13-cis and all-trans bR.