Transient absorption and photovoltage study of' self-assembled bacteriorhodopsin/polycation multilayer films

A series of organized (PDAC/PM)(n) (poly(diallyldimethylammonium chloride)/purple membrane) multilayer films were prepared by alternate adsorptions of positively charged PDAC polyelectrolyte and negatively charged purple membrane (PM). The kinetics of the photocycle of bacteriorhodopsin (bR) in PM was studied by flash photolysis and transient photovoltage methods. Although the orientation of the adsorbed bR depends on the pH of the PM suspension, the kinetics of the photo-induced reaction cycle in dehydrated films is independent of the deposition pH. In dry (PDAC/PM)(n) films the decay of the M intermediate to the initial bR state is multiexponential and delayed to several minutes for both orientations. A simultaneous two-exponential decay in millisecond time domain was observed at red wavelengths. The source of the red-shifted absorption is suggested to be the C(610) intermediate of the cis photocycle of bR.     

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.     

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.     

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.     

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.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Effect of iodination of the purple membrane on the photocycle of bacteriorhodopsin

Bacteriorhodopsin in the purple membrane of Halobacterium halobium is coupled to a photocycle that results in the release and uptake of protons. The role of tyrosyl residues in the photocycle of bacteriorhodopsin has been investigated by the technique of chemical modifications of these residues by iodination and nitration. The studies indicate that modification of a tyrosyl residue accelerates M₄₁₂ formation, whereas modification of another type of tyrosine residue(s) accessible from the cytoplasmic surface of the purple membrane inhibits M₄₁₂ decay. The results support the hypothesis that a reversible deprotonation of tyrosine residues prior to and after M₄₁₂ formation in the photocycle are steps in the light-driven pathway of H⁺ translocation by bacteriorhodopsin.     

Substitution of membrane-embedded aspartic acids in bacteriorhodopsin causes specific changes in different steps of the photochemical cycle

Millisecond photocycle kinetics were measured at room temperature for 13 site-specific bacteriorhodopsin mutants in which single aspartic acid residues were replaced by asparagine, glutamic acid, or alanine. Replacement of aspartic acid residues expected to be within the membrane-embedded region of the protein (Asp-85, -96, -115, or -212) produced large alterations in the photocycle. Substitution of Asp-85 or Asp-212 by Asn altered or blocked formation of the M410 photointermediate. Substitution of these two residues by Glu decreased the amount of M410 formed. Substitutions of Asp-96 slowed the decay rate of the M410 photointermediate, and substitutions of Asp-115 slowed the decay rate of the O640 photointermediate. Corresponding substitutions of aspartic acid residues expected to be in cytoplasmic loop regions of the protein (Asp-36, -38, -102, or -104) resulted in little or no alteration of the photocycle. Our results indicate that the defects in proton pumping which we have previously observed upon substitution of Asp-85, Asp-96, Asp-115, and Asp-212 [Mogi, T., Stern, L. J., Marti, T., Chao, B. H., & Khorana, H. G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4148-4152] are closely coupled to alterations in the photocycle. The photocycle alterations observed in these mutants are discussed in relation to the functional roles of specific aspartic acid residues at different stages of the bacteriorhodopsin photocycle and the proton pumping mechanism.     

Structural determinants of purple membrane assembly

The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of Halobacterium salinarum. High-resolution structural studies, in conjunction with detailed knowledge of the lipid composition, make the purple membrane one of the best models for elucidating the forces that are responsible for the assembly and stability of integral membrane protein complexes. In this review, recent mutational efforts to identify the structural features of bacteriorhodopsin that determine its assembly in the purple membrane are discussed in the context of structural, calorimetric and reconstitution studies. Quantitative evidence is presented that interactions between transmembrane helices of neighboring bacteriorhodopsin molecules contribute to purple membrane assembly. However, other specific interactions, particularly between bacteriorhodopsin and lipid molecules, may provide the major driving force for assembly. Elucidating the molecular basis of protein-protein and protein-lipid interactions in the purple membrane may provide insights into the formation of integral membrane protein complexes in other systems.     

Photoconversion from the light-adapted to the dark-adapted state of bacteriorhodopsin.

Dark and light adaptation of bacteriorhodopsin in purple membrane multilayers at less than 100% relative humidity differs from that seen in suspensions. Equilibrium between the two bacteriorhodopsin isomers (bR cis 550 and bR trans 570) in the light-adapted state becomes dependent on the wavelength of actinic light. Excitation at the red edge of the visible absorption band causes dark adaptation in a light-adapted sample. Using polarized actinic and measuring light, we show that acceleration of the dark adaptation through heating by actinic light cannot explain this observation. A light-driven bR trans 570 to bR cis 550 reaction that competes with the well-known 13 cis-to-all-trans light adaptation reaction must exist under our experimental conditions. Trans-to-cis conversion is a one-photon process distinct from the two photon process observed by others in purple membrane suspensions (Sperling, W., C. N. Rafferty, K. D. Kohl, and N. A. Dencher, 1978, FEBS (Fed. Eur. Biochem. Soc.) Lett. 97:129-132). Its quantum efficiency increases monotonously on reducing the hydration level, and is paralleled by an increase in the lifetime of the M410 intermediate of the trans photocycle. We suggest that at this point a branch leads from the all-trans into the 13-cis photocycle. It is probably the same reaction that causes the reduced light adaptation in monomeric bacteriorhodopsin (Casadio, R., H. Gutowitz, P. Mowery, M. Taylor, and W. Stoeckenius, 1980, Biochim. Biophys. Acta. 590:13-23; Casadio, R., and W. Stoeckenius, 1980, Biochemistry. 19:3374-3381).     

Effects of pressure and temperature on the M412 intermediate of the bacteriorhodopsin photocycle. Implications for the phase transition of the purple membrane

The effects of pressure and temperature on the decay kinetics of the M412 (M) intermediate in the photocycle of bacteriorhodopsin were studied to provide information about the phase transitions of the purple membrane lipids. The activation volume (delta V++) for the decay of M is expected to be different below and above a phase transition. However, no abrupt change in delta V++ was found from 3.5 degrees to 60 degrees C. But a sharp break was observed in a plot of the logarithm of the rate of M decay vs. pressure. Extrapolation of this break point to standard atmospheric pressure gives a temperature of -42 degrees C, which probably corresponds to the phase transition of the purple membrane lipids. This conclusion is supported by studies of the effect of pressure on the M kinetics of bacteriorhodopsin incorporated into dimyristoylphosphatidylcholine vesicles, whose phase transition has previously been characterized.     

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.     

Beta-amyloid 25 to 35 is intercalated in anionic and zwitterionic lipid membranes to different extents

D96N bacteriorhodopsin has two photointermediates with the deprotonated Schiff base: the M and MN intermediates. We measure the time-resolved x-ray diffraction of the D96N purple membrane after flash photoexcitation (pH 7.0, 25 degrees C). The data clearly show the M-MN transition during the D96N photocycle. Low-resolution projection maps of these states show that the F helix of the MN intermediate shifts from its original position and this shift is much larger than that of the M intermediate. This indicates that the F helix moves in the M-MN transition of the D96N bacteriorhodopsin photocycle. Moreover, the existence of the MN intermediate in the D96N photocycle under neutral pH indicates that the MN intermediate is not peculiar to the alkaline condition. It is notable that the structural transition of M-MN is independent of the protonation state of the Schiff base. Therefore, the F helix movement precedes reprotonation of the Schiff base in the bacteriorhodopsin photocycle. Our previous study showed that the M-MN transition is hydration-dependent and that the MN intermediate is more hydrated than the M intermediate. Considering this together with the present results, we conclude that the movement of the F helix causes hydration of the cytoplasmic side, which promotes the reprotonation of the Schiff base.     

Regulation of the bacteriorhodopsin photocycle and proton pumping in whole cells of Halobacterium salinarium.

Single-turnover kinetics of the bacteriorhodopsin photocycle and proton-pumping capabilities of whole cells were studied. It was found that the Delta mu (tilde)H+ of the cell had a profound influence on the kinetics and components of the cycle. For example, comparing the photocycle in whole cells to that seen in PM preparations, we found that (1) the single-turnover time of the cycle was increased approximately 10-fold, (2) the mole fraction of M-fast (at high actinic light) decreased from 50 to 20%, and (3) the time constant for M-slow increased significantly. The level of Delta mu(tilde)H+ was dependent on respiration, ATP formation and breakdown, and the magnitude of a pre-existing K+ diffusion gradient. The size of the Delta mu(tilde)H+ could be manipulated by additions of HCN, nigericin, and DCCD (N,N'-dicyclohexylcarbodamide). At higher levels of Delta mu(tilde)H+, further changes in the photocycle were seen. (4) Two slower components of M-decay appeared as major components. (5) The apparent conversion of the M-fast to the O intermediate disappeared. (6) A partial reversal of an early photocycle step occurred. The photocycle of intact cells could be changed to that seen in purple membrane suspensions by the energy-uncoupler CCCP or by lysis of the cells. In fresh whole cells, light-induced proton pumping was not seen until the K+ diffusion potential was dissipated and proton accumulation facilitated by use of a K+-H+ exchanger (nigericin), respiration was inhibited by HCN, and ATP synthesis and breakdown were inhibited by DCCD. In stored cells, the pre-existing K+ diffusion gradient was diminished through slow diffusion, and only DCCD and HCN were required to elicit proton extrusion.     

Photoreactions of bacteriorhodopsin at acid pH.

It has been known that bacteriorhodopsin, the retinal protein in purple membrane which functions as a light-driven proton pump, undergoes reversible spectroscopic changes at acid pH. The absorption spectra of various bacteriorhodopsin species were estimated from measured spectra of the mixtures that form at low pH, in the presence of sulfate and chloride. The dependency of these on pH and the concentration of Cl- fit a model in which progressive protonation of purple membrane produces "blue membrane", which will bind, with increasing affinity as the pH is lowered, chloride ions to produce "acid purple membrane." Transient spectroscopy with a multichannel analyzer identified the intermediates of the photocycles of these altered pigments, and described their kinetics. Blue membrane produced red-shifted KL-like and L-like products, but no other photointermediates, consistent with earlier suggestions. Unlike others, however, we found that acid purple membrane exhibited a very different photocycle: its first detected intermediate was not like KL in that it was much more red-shifted, and the only other intermediate detectable resembled the O species of the bacteriorhodopsin photocycle. An M-like intermediate, with a deprotonated Schiff base, was not found in either of these photocycles. There are remarkable similarities between the photoreactions of the acid forms of bacteriorhodopsin and the chloride transport system halorhodopsin, where the Schiff base deprotonation seems to be prevented by lack of suitable aspartate residues, rather than by low pH.     

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.     

Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer

A one-of-a-kind high speed optical multichannel spectrometer was designed and built at NIH and described in this journal in 1997 [J.W. Cole, R.W. Hendler, P.D. Smith, H.A. Fredrickson, T.J. Pohida, W.S. Friauf. A high speed optical multichannel analyzer. J Biochem Biophys Methods 1997;35:16–174.]. The most unique aspect of this instrument was the ability to follow an entire time course from a single activation using a single sample. The instrument has been used to study rapid kinetic processes in the photon-driven bacteriorhodopsin photocycle and electron transport from cytochrome c to cytochrome aa₃ and from cytochrome aa₃ to oxygen. The present paper describes a second generation instrument with a number of important enhancements which significantly improve its capabilities for multichannel kinetic studies. An example application is presented in which the kinetics of photon-induced proton flow across the biological membrane is measured simultaneously with the individual steps of the photocycle determined optically. Matching the time constants for the two processes indicates which molecular transformations are associated with major proton movements.