Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique

Thin films of poly(allylamine hydrochloride) (PAH) and bacteriorhodopsin (bR) embedded in purple membrane (PM) have been prepared by layer-by-layer (LBL) self-assembly technique. The results obtained by UV-Vis spectroscopy and atomic force microscopy (AFM) analysis showed that the biological activity of bR was preserved and PM fragments could be well oriented onto the ITO substrate. A photo-electrochemical cell with the structure of ITO/(PAH/PM)(n)/electrolyte (0.5M KCl)/Pt was fabricated and studied. The photocurrent peaks of (PAH/PM)(6) corresponding to light-on and light-off were about 200 and 100 nA/cm(2), respectively, with the former enhanced 30% higher than that of the reference films made of (PDAC/PM)(6).     

The Photocycle of Bacteriorhodopsin in the Two-Dimensional Orthorombic Crystal Form of Purple Membrane

Laser flash photolysis and low-temperature absorption studies of the photocycle of orthorhombic purple membrane (o-PM) reveal the existence of the same K, L, and M intermediates as found in the native hexagonal purple membrane (h-PM). However, the 0 intermediate is missing in the o-PM. The absorption spectrum of the K intermediate of o-PM is blueshifted by ~15 nm relative to the K intermediate found in the hexagonal purple membrane. The decay relaxation time constants of M in the o-PM are higher by more than an order of magnitude than the corresponding relaxation time constants in the h-PM. Similarly to the h-PM, the decay of M depends on the pulse width of excitation. The time-independent anisotropy factor obtained in photoselection studies of the M intermediate demonstrates the complete immobility of bacteriorhodopsin (bR) within the o-PM matrix. The same anisotropy factor of 0.3 obtained for o-PM and for h-PM suggests that in both crystalline lattices the transition moment of the retinal chromophore has similar angles with the plane of the membrane. The dependence of the decay kinetics of M on its occupancy may suggest the existence of kinetic coupling between neighboring bR molecules.     

Photoacoustic photocalorimetry and spectroscopy of Halobacterium halobium purple membranes.

Enthalpy changes associated with intermediates of the photocycle of bacteriorhodopsin (bR) in light-adapted Halobacterium halobium purple membranes, and decay times of these intermediates, are obtained from photoacoustic measurements on purple membrane fragments. Our results, mainly derived from modulation frequency spectra, show changes in the amount of energy stored in the intermediates and in their decay times as a function of pH and/or salt concentration. Especially affected are the slowest step (endothermic) and a spectroscopically unidentified intermediate (both at pH 7). This effect is interpreted in terms of cation binding to the protein, conformational changes of which are thought to be connected with the endothermic process. Wavelength spectra are used to obtain heat dissipation spectra, which allow identification of wavelength regions with varying photoactivity, and estimation of the amounts of enthalpy stored in the photointermediates. Because of bleaching and accumulation of intermediates, however, and because of the small fraction of light energy stored during photocycle, quantitative information cannot be obtained. From photoacoustic wavelength spectra of purple membrane fragments equilibrated at 63% relative humidity, rise and decay times of the bR570 and M412 intermediates are calculated.     

CHAPS对M_(412)的动力学和共振拉曼光谱的影响

研究了表面活性剂CHAPS对紫膜中菌紫质在光循环过程中M_(412)的衰减速率及质子泵功能的影响.光循环中间体M_(412)快、慢衰减组分的衰减速率及质子衰减速率均受到CHAPS的影响,综合激光拉曼光谱对M_(412)和其它光循环中间体相对含量的测定,表明CHAPS对紫膜的影响是通过影响其膜脂完整的液晶结构,而使紫膜光循环动力学过程及质子泵功能发生变化的.     

Roles of functional lipids in bacteriorhodopsin photocycle in various delipidated purple membranes

Purple membrane (PM) is composed of several native lipids and the transmembrane protein bacteriorhodopsin (bR) in trimeric configuration. The delipidated PM (dPM) samples can be prepared by treating PM with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) to partially remove native lipids while maintaining bR in the trimeric configuration. By correlating the photocycle kinetics of bR and the exact lipid compositions of the various dPM samples, one can reveal the roles of native PM lipids. However, it is challenging to compare the lipid compositions of the various dPM samples quantitatively. Here, we utilize the absorbances of extracted retinal at 382 nm to normalize the concentrations of the remaining lipids in each dPM sample, which were then quantified by mass spectrometry, allowing us to compare the lipid compositions of different samples in a quantitative manner. The corresponding photocycle kinetics of bR were probed by transient difference absorption spectroscopy. We found that the removal rate of the polar lipids follows the order of BPG ≈ GlyC < S-TGD-1 ≈ PG < PGP-Me ≈ PGS. Since BPG and GlyC have more nonpolar phytanyl groups than other lipids at the hydrophobic tail, causing a higher affinity with the hydrophobic surface of bR, the corresponding removal rates are slowest. In addition, as the reaction period of PM and CHAPS increases, the residual amounts of PGS and PGP-Me significantly decrease, in concomitance with the decelerated rates of the recovery of ground state and the decay of intermediate M, and the reduced transient population of intermediate O. PGS and PGP-Me are the lipids with the highest correlation to the photocycle activity among the six polar lipids of PM. From a practical viewpoint, combining optical spectroscopy and mass spectrometry appears a promising approach to simultaneously track the functions and the concomitant active components in a given biological system.     

M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH

The dependence of the bacteriorhodopsin (bR) photocycle on the intensity of the exciting flash was investigated in purple membranes. The dependence was most pronounced at slightly alkaline pH values. A comparison study of the kinetics of the photocycle and proton uptake at different intensities of the flash suggested that there exist two parallel photocycles in purple membranes at a high intensity of the flash. The photocycle of excited bR in a trimer with the two other bR molecules nonexcited is characterized by an almost irreversible M --> N transition. Excitation of two or three bR in a trimer induces the N --> M back reaction and accelerates the N --> bR transition. Based on the qualitative similarity of the pH dependencies of the photocycles of solubilized bR and excited dimers and trimers we proposed that the interaction of nonexcited bR in trimers alters the photocycle of the excited monomer as compared to solubilized bR and the changes in the photocycles in excited dimers and trimers are the result of decoupling of this interaction.     

Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity.

Specific lipids of the purple membrane of Halobacteria are required for normal bacteriorhodopsin structure, function, and photocycle kinetics [Hendler, R.W. & Dracheva, S. (2001) Biochemistry (Moscow)66, 1623-1627]. The decay of the M-fast intermediate through a path including the O intermediate requires the presence of a hydrophobic environment near four charged aspartic acid residues within the cytoplasmic loop region of the protein (R. W. Hendler & S. Bose, unpublished results). On the basis of the unique ability of squalene, the most hydrophobic purple membrane lipid, to induce recovery of M-fast activity in Triton-treated purple membrane, we proposed that this uncharged lipid modulates an electrostatic repulsion between the membrane surface of the inner trimer space and the nearby charged aspartic acids of the cytoplasmic loop region to promote transmembrane alpha-helical mobility with a concomitant increase in the speed of the photocycle. We examined Triton-treated purple membranes in various stages of reconstitution with native lipid suspensions using infrared spectroscopic techniques. We demonstrate a correlation between the vibrational half-width parameter of the protein alpha-helical amide I mode at 1660 cm-1, reflecting the motional characteristics of the transmembrane helices, and the lipid-induced recovery of native bacteriorhodopsin properties in terms of the visible absorbance maxima of ground state bacteriorhodopsin and the mean decay times of the photocycle M-state intermediates.     

Effect of anions on the photocycle of halorhodopsin. Substitution of chloride with formate anion

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 NO(2)(-) 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.     

Tuning the Photocycle Kinetics of Bacteriorhodopsin in Lipid Nanodiscs

Monodisperse lipid nanodiscs are particularly suitable for characterizing membrane protein in near-native environment. To study the lipid-composition dependence of photocycle kinetics of bacteriorhodopsin (bR), transient absorption spectroscopy was utilized to monitor the evolution of the photocycle intermediates of bR reconstituted in nanodiscs composed of different ratios of the zwitterionic lipid (DMPC, dimyristoyl phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine) to the negatively charged lipid (DOPG, dioleoyl phosphatidylglycerol; DMPG, dimyristoyl phosphatidylglycerol). The characterization of ion-exchange chromatography showed that the negative surface charge of nanodiscs increased as the content of DOPG or DMPG was increased. The steady-state absorption contours of the light-adapted monomeric bR in nanodiscs composed of different lipid ratios exhibited highly similar absorption features of the retinal moiety at 560 nm, referring to the conservation of the tertiary structure of bR in nanodiscs of different lipid compositions. In addition, transient absorption contours showed that the photocycle kinetics of bR was significantly retarded and the transient populations of intermediates N and O were decreased as the content of DMPG or DOPG was reduced. This observation could be attributed to the negatively charged lipid heads of DMPG and DOPG, exhibiting similar proton relay capability as the native phosphatidylglycerol (PG) analog lipids in the purple membrane. In this work, we not only demonstrated the usefulness of nanodiscs as a membrane-mimicking system, but also showed that the surrounding lipids play a crucial role in altering the biological functions, e.g., the ion translocation kinetics of the transmembrane proteins.     

Photoinduced kinetics of bacteriorhodopsin in a dried xerogel glass

Time-resolved absorption measurements of the formation and decay kinetics of the M (deprotonated) photocycle intermediate of bR purple membranes entrapped within a dried xerogel glass have been investigated. The dramatic change observed for the M state decay time is in contrast to the relatively insensitive half life reported for the M intermediate of the D96N mutant entrapped within a dried sol-gel glass. The decay kinetics of the M intermediate was observed to slow by a factor of almost 100 when the solvent was removed from the wet-gel to form the dry xerogel glass. Very long aging times for wet-gels resulted in highly biexponential M state decay kinetics. Upon drying, the M state formation rate initially decreased relative to that in solution before increasing in the dry xerogel to a formation rate nearly three times faster than in solution.     

Effects of detergent environments on the photocycle of purified monomeric bacteriorhodopsin

Time-resolved difference spectra have been obtained for the photocycle of delipidated bacteriorhodopsin monomers (d-BR) in six different detergent micelle environments that were prepared by two new detergent-exchange techniques. A global kinetic analysis of the photocycle spectra for d-BR in each detergent environment was performed. Comparison of these results with those obtained for the photocycle of bacteriorhodopsin in purple membrane (PM) shows that there is one fewer kinetically distinguishable process for monomeric BR between the decay of the K intermediate and the rise of the M intermediate. Assuming a sequential pathway occurs in the photocycle, it appears that the equilibrium between the L and M intermediates is reached much more rapidly in the detergent micelles. This is attributed to a more direct interaction between Asp-85 and the proton on the nitrogen of the Schiff base of retinal for BR in the detergents. Equilibrium concentrations of late photocycle intermediates are also altered in detergents. The later steps of the photocycle, including the decay of the M intermediate, are slowed in detergents with rings in their hydrocarbon region. This is attributed to effects on conformational changes occurring during the decay of M and/or other later photocycle intermediates. The lifetime of dark adaptation of light-adapted d-BR in different detergent environments increases in environments where the lifetime of the M intermediate increases. These results suggest that the high percentage of either unsaturated or methyl-branched lipids in PM and the membranes of other retinal proteins may be important for their effective functioning.     

Time-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics

The determination of the intermediate state structures of the bacteriorhodopsin photocycle has lead to an unprecedented level of understanding of the catalytic process exerted by a membrane protein. However, the crystallographic structures of the intermediate states are only relevant if the working cycle is not impaired by the crystal lattice. Therefore, we applied visible and Fourier transform infrared spectroscopy (FTIR) microspectroscopy with microsecond time resolution to compare the photoreaction of a single bacteriorhodopsin crystal to that of bacteriorhodopsin residing in the native purple membrane. The analysis of the FTIR difference spectra of the resolved intermediate states reveals great similarity in structural changes taking place in the crystal and in PM. However, the kinetics of the photocycle are significantly altered in the three-dimensional crystal as compared to PM. Strikingly, the L state decay is accelerated in the crystal, whereas the M decay is delayed. The physical origin of this deviation and the implications for trapping of intermediate states are discussed. As a methodological advance, time-resolved step-scan FTIR spectroscopy on a single protein crystal is demonstrated for the first time which may be used in the future to gauge the functionality of other crystallized proteins with the molecular resolution of vibrational spectroscopy.     

Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin

The effect of lipid-protein interaction on the photodynamics of bacteriorhodopsin (bR) was investigated by using partially delipidated purple membrane (pm). When pm was incubated with a mild detergent, Tween 20, the two major lipid components of pm, phospholipids and glycolipids, were released in different ways: the amount of phospholipids released was proportional to the logarithm of the incubation time; the release of glycolipids became noticeable after the release of approximately 2 phospholipids/bR, but soon leveled off at approximately 50% of the initial content. It was found that the thermal decay of the photocycle intermediate N560 was inhibited by the removal of less than 2 phospholipids per bR. This inhibition was partly explained by an increase in the local pH near the membrane surface. More significant changes in the bR photoreactions were observed when greater than 2 phospholipids/bR were removed: (1) the extent of light adaptation became much smaller, and this reduction correlated with the release of glycolipids; (2) N560 became difficult to detect; (3) the M412 intermediate, which is characterized by a pH-insensitive lifetime, was replaced by a long-lived M-like photoproduct with a pH-sensitive lifetime. The heavy delipidation apparently altered the mechanism by which the deprotonated Schiff base receives a proton. An important conformational change in the protein moiety is suggested to take place during the M412 state, this conformational change being inhibited in the rigid lipid environment.     

血卟啉衍生物（HPD）对紫膜菌紫质（bR）的光敏化作用

研究了血卟啉衍生物（ＨＰＤ）对嗜盐菌紫膜上蛋白质菌紫质（ｂＲ）的光敏化作用,结果表明,ＨＰＤ与紫膜结合并不影响ｂＲ的光学性质及活性;但经光照射、ＨＰＤ光敏反应后,ｂＲ丧失光循环活性。进一步的探测显示ｂＲ中的视黄醛色素团及色氨酸均在光敏反应中受损,反映了除视黄醛色素团有可能直接受损外,深埋于折叠蛋白内部的部分色氨酸残基。亦可能在ＨＰＤ光敏化过程中被损伤。实验证明,单线态氧（＾１Ｏ２）的作用是ＨＰＤ光     

Proton transfer reactions in native and deionized bacteriorhodopsin upon delipidation and monomerization

We have investigated the role of the native lipids on bacteriorhodopsin (bR) proton transfer and their connection with the cation-binding role. We observe that both the efficiency of M formation and the kinetics of M rise and decay depend on the lipids and lattice but, as the lipids are removed, the cation binding is a much less important factor for the proton pumping function. Upon 75% delipidation using 3-[(cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS), the M formation and decay kinetics are much slower than the native, and the efficiency of M formation is approximately 30%-40% that of the native. Upon monomerization of bR by Trition X-100, the efficiency of M recovers close to that of the native (depending on pH), M formation is approximately 10 times faster, and M decay kinetics are comparable to native at pH 7. The same results on the M intermediate are observed if deionized blue bR (deI bbR) is treated with these detergents (with or without pH buffers present), even though deionized blue bR containing all the lipids has no photocycle. This suggests that the cation(s) has a role in native bR that is different than in delipidated or monomerized bR, even so far as to suggest that the cation(s) becomes unimportant to the function as the lipids are removed.     

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the protein residues that control the yield and kinetics of O(630) in the photocycle of bacteriorhodopsin

The effects of pH on the yield (phi(r)), and on the apparent rise and decay constants (k(r), k(d)), of the O(630) intermediate are important features of the bacteriorhodopsin (bR) photocycle. The effects are associated with three titration-like transitions: 1) A drop in k(r), k(d), and phi(r) at high pH [pK(a)(1) approximately 8]; 2) A rise in phi(r) at low pH [pK(a)(2) approximately 4.5]; and 3) A drop in k(r) and k(d) at low pH [pK(a)(3) approximately 4. 5]. (pK(a) values are for native bR in 100 mM NaCl). Clarification of these effects is approached by studying the pH dependence of phi(r), k(r), and k(d) in native and acetylated bR, and in its D96N and R82Q mutants. The D96N experiments were carried out in the presence of small amounts of the weak acids, azide, nitrite, and thiocyanate. Analysis of the mutant's data leads to the identification of the protein residue (R(1)) whose state of protonation controls the magnitude of phi(r), k(r), and k(d) at high pH, as Asp-96. Acetylation of bR modifies the Lys-129 residue, which is known to affect the pK(a) of the group (XH), which releases the proton to the membrane exterior during the photocycle. The effects of acetylation on the O(630) parameters reveal that the low-pH titrations should be ascribed to two additional protein residues R(2) and R(3). R(2) affects the rise of phi(r) at low pH, whereas the state of protonation of R(3) affects both k(r) and k(d). Our data confirm a previous suggestion that R(3) should be identified as the proton release moiety (XH). A clear identification of R(2), including its possible identity with R(3), remains open.     

Large transient nonproton ion movements in purple membrane suspensions are abolished by solubilization in Triton X-100.

Light-induced release/uptake of both protons and other ions cause transient changes in conductivity in suspensions of purple membrane (PM) fragments (Marinetti, Tim, and David Mauzerall, 1983, Proc. Natl. Acad. Sci. USA, 80:178-180). We find that the release/uptake of nonproton ions with quantum yield greater than 1 is observed at most pHs and ionic strengths. Only at both low pH and low ionic strength is the conductivity transient mostly due to protons. Our hypothesis is that during the photocycle, changes occur in the PM's dense surface charge distribution that result in changes in the number of counterions bound or condensed at the membrane surface. To test this, the PM structure was perturbed with the nonionic detergent Triton X-100. Immediately after addition, Triton does not abolish the nonproton ion movements; in fact at low detergent concentrations (0.02% vol/vol) the signal amplitudes increased considerably. However, when PM is completely solubilized into monomers in Triton, the conductivity transients are due to protons alone, though at lower quantum yield compared with native PM. These results suggest that changes in the surface charge distribution in native PM's photocycle could contribute to proton transfer between the aqueous phase and bR itself.     

Phase-lifetime spectrophotometry of deoxycholate-purified bacteriorhodopsin reconstituted into asolectin vesicles

A rapid reconstitution procedure has been developed to insert deoxycholate-purified bacteriorhodopsin (bR) into asolectin vesicles. The procedure relies on the ability of the hydrophobic resin Bio-Beads SM-2 to remove octyl glucoside from a mixture of deoxycholate-purified bR, asolectin, and the detergent. Light-dependent acidification of the vesicle interior is observed with the reconstituted preparations as judged by the fluorescence quenching of an entrapped pH indicator, pyranine. Inhibition of proton pumping by the addition of LaCl3 to the external medium indicates that approximately 90% of the bR is oriented such that it pumps protons into the vesicles. Phase-lifetime spectrophotometry was used to study the relaxation processes associated with the intermediate in the photocycle of the reconstituted bR which absorbs at 410 nm. Amplitude spectra indicate that these absorbance changes are associated with the M intermediate in the bR photocycle. Two relaxation processes are observed. One is characterized by a relaxation time of approximately 4 ms and is independent of pH over the range 4.4-9.4. The longer relaxation time varies from 4 to 200 ms in the same pH range. By digitization of transients, which are observable when the actinic source is modulated at a low frequency, information about the dependence of the slower process on the light intensity and carbonyl cyanide m-chlorophenylhydrazone was obtained. The results can be interpreted in terms of two different forms of the M intermediate that decay on parallel kinetic paths. To explain the pH dependence of the decay rate, the slower decaying form must have three coupled protonation states, each with a different decay rate.