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.     

The residues Leu 93 and Asp 96 act independently in the bacteriorhodopsin photocycle: studies with the leu 93-->Ala, Asp 96-->Asn double mutant.

Previous mutagenesis studies with bacteriorhodopsin have shown that reprotonation of the Schiff's base is the rate-limiting step in the photocycle of the D96N mutant, whereas retinal re-isomerization and return of the protein to the initial state constitute the rate-limiting events in the photocycle of the L93A mutant. Thus, in the D96N mutant, decay of the M intermediate is slowed down by more than 100-fold at pH 7. In the L93A mutant, decay of the O intermediate is slowed down by 250-fold. We report here that in the L93A, D96N double mutant, decay of the M intermediate, as well as the formation and decay of the O intermediate, are slowed down dramatically. The photocycle is completed by the decay of a long-lived O intermediate, as in the L93A mutant. The decay of the M and O intermediates in the double mutant parallels the behavior seen in the single mutants over a wide temperature and pH range, arguing that the observed independence is an intrinsic property of the mutant. The slow decay of the M and O intermediates can be selectively and independently reversed under conditions identical to those used for the corresponding intermediates in the D96N and L93A single mutants. Because the effects of the two individual mutations are preserved in the double mutant and can be independently reversed, we conclude that residues Asp 96 and Leu 93 act independently and at different stages of the bacteriorhodopsin photocycle. These results also show that formation of the O intermediate only requires protonation of the Schiff's base and is independent of the protonation of Asp 96 from the aqueous medium.     

Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin

The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.     

Photocycle of dried acid purple form of bacteriorhodopsin.

The photocycle of dried bacteriorhodopsin, pretreated in a 0.3 M HCl solution, was studied. Some properties of this dried sample resemble that of the acid purple suspension: the retinal conformation is mostly all-trans, 15-anti form, the spectrum of the sample is blue-shifted by 5 nm to 560 nm, and it has a truncated photocycle. After photoexcitation, a K-like red-shifted intermediate appears, which decays to the ground state through several intermediates with spectra between the K and the ground state. There are no other bacteriorhodopsin-like intermediates (L, M, N, O) present in the photocycle. The K to K' transition proceeds with an enthalpy decrease, whereas during all the following steps, the entropic energy of the system decreases. The electric response signal of the oriented sample has only negative components, which relaxes to zero. These suggest that the steps after intermediate K represent a relaxation process, during which the absorbed energy is dissipated and the protein returns to its original ground state. The initial charge separation on the retinal is followed by limited charge rearrangements in the protein, and later, all these relax. The decay times of the intermediates are strongly influenced by the humidity of the sample. Double-flash experiments proved that all the intermediates are directly driven back to the ground state. The study of the dried acid purple samples could help in understanding the fast primary processes of the protein function. It may also have importance in technical applications.     

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.     

Spectroscopic evidence for the formation of an N intermediate during the photocycle of sensory rhodopsin II (phoborhodopsin) from Natronobacterium pharaonis

Sensory rhodopsin II is a seven transmembrane helical retinal protein and functions as a photoreceptor protein in negative phototaxis of halophilic archaea. Sensory rhodopsin II from Natronomonas pharaonis (NpSRII) is stable under various conditions and can be expressed functionally in Escherichia coli cell membranes. Rhodopsins from microorganisms, known as microbial rhodopsins, exhibit a photocycle, and light irradiation of these molecules leads to a high-energy intermediate, which relaxes thermally to the original pigment after passing through several intermediates. For bacteriorhodopsin (BR), a light-driven proton pump, the photocycle is established as BR → K → L → M → N → O → BR. The photocycle of NpSRII is similar to that of BR except for N, i.e., M thermally decays into the O, and N has not been well characterized in the photocycle. Thus we here examined the second half of the photocycle in NpSRII, and in the present transient absorption study we found the formation of a new photointermediate whose absorption maximum is ～500 nm. This intermediate becomes pronounced in the presence of azide, which accelerates the decay of M. Transient resonance Raman spectroscopy was further applied to demonstrate that this intermediate contains a 13-cis retinal protonated Schiff base. However, detailed analysis of the transient absorption data indicated that M-decay does not directly produce N but rather produces O that is in equilibrium with N. These observations allowed us to propose a structural model for a photocycle that involves N.     

Transient spectroscopy of bacterial rhodopsins with an optical multichannel analyzer. 1. Comparison of the photocycles of bacteriorhodopsin and halorhodopsin

We used a gated optical multichannel analyzer to measure transient flash-induced absorption changes in bacteriorhodopsin (BR) and halorhodopsin (HR) and developed criteria for calculating the absorption spectra of the photocycle intermediates and the kinetics of their rise and decay. The results for BR agree with data reported by a large number of other authors. The results for HR in the presence of chloride are consistent with earlier data and reveal an additional intermediate, not previously seen, in the submicrosecond time scale. Although an M412-like intermediate is not in the HR photocycle, a one-by-one comparison of the rest of the intermediates observed for BR and HR indicates a striking similarity between the photocycles of the two bacterial rhodopsins. This was previously not apparent, perhaps because the experimental approaches to the spectroscopy of the two pigments were different and the data were thus more fragmented.     

Activation parameters for the halorhodopsin photocycle: a phase lifetime spectroscopic study of the 520- and 640-nanometer intermediates

Phase lifetime spectroscopy is used to investigate the kinetics of the 520- and 640-nm intermediates in the halorhodopsin photocycle. These intermediates decay on the millisecond time scale and are strongly implicated in the chloride transport steps. The temperature dependence of the 520 and 640 relaxations was measured for chloride and nitrate buffers at pH 6, 7, and 8 and for iodide buffer at pH 6. The 640 relaxations have small activation energies but large entropy barriers. The two relaxation times observed for the 640 intermediate were interpreted by using a mechanism in which two 640 species exist in equilibrium. The second 640 species is not along the main decay path for the photocycle. A quantitative analysis of the data allowed rate constants and activation parameters to be calculated for the elementary steps of this isomerization process. These parameters are similar for both chloride and nitrate buffers but differ somewhat in iodide. The derived calculated rate constants were consistent with the relaxation times observed for the 520 intermediate. These results indicate that the 520 and two 640 intermediates have very similar free energies as well as similar free energies of activation for the various interconversion processes.     

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.     

Roles of the D-ribose and 5,6-dimethylbenzimidazole moieties of the nucleotide loop of adenosylcobalamin in manifestation of coenzymic function in the diol dehydrase reaction

Coenzyme analogs in which the D-ribose moiety of the nucleotide loop was replaced by an oligomethylene group and a trimethylene analog containing imidazole instead of 5,6-dimethylbenzimidazole were synthesized. Coordination of the 5,6-dimethylbenzimidazole to the cobalt atom in these analogs was much weaker than that in cobalamins. The replacement of this base with imidazole did not significantly alter the strength of the coordination to the cobalt atom. 5,6-Dimethylbenzimidazolyl trimethylene and tetramethylene and imidazolyl trimethylene analogs were partially active as coenzymes in the diol dehydrase reaction in this order as judged by kcat, but the others were not active as coenzymes and were weak competitive inhibitors. This indicates that neither the alpha-D-ribofuranose ring nor the functional groups of the ribose moiety are essential for coenzymic function. There was an optimum loop size of the analogs for catalysis and for tight binding to the apoenzyme, which corresponds to the loop size of cobalamins. Therefore, the D-ribose moiety seems important as a spacer to keep the base in the proper position. The reaction with the imidazolyl trimethylene analog as coenzyme was accompanied with concomitant rapid inactivation during catalysis. The inactivation occurred only in the presence of substrate. Upon inactivation with this analog, 5'-deoxyadenosine and a B12r-like species were formed from the adenosyl group and the rest of the analog molecule, respectively, without modification of the apoenzyme. Therefore, it can be concluded that this is a kind of suicide inactivation which occurred from one of the intermediates in the normal catalytic process. The dimethylbenzo moiety of the regular coenzyme thus seems to play an important role in preventing the intermediate complexes from inactivation during catalysis.     

Characterization of the proton-transporting photocycle of pharaonis halorhodopsin

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.     

两种状态细菌视紫红质光循环中间产物与pH的关系

本文主要用微机控制的毫秒级闪光动力学光谱仪研究含三体细菌视紫红质(Bacteriorhodopsin,简称BR)的紫膜碎片和含单体BR的DMPC(dimyristoyl-Phosphatidyl-choline)脂质囊泡在不同pH条件下光循环中间产物M_(412)和O_(640)的变化,研究结果表明:BR单体与其三体状态相比,BR单体的光循环中间产物M_(412)的产量受介质pH变化的影响较大,其慢衰减成份的衰减比三体BR慢3—10倍.说明单体BR的结构状态较易受PH影响,单体BR光循环中间产物O_(640)随pH变化的趋势与三体BR的有很大区别,可能是由于不同状态的BR受pH的影响,但其具有不同的构型,导致光循环途径的变化.     

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.     

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.     

Synthesis and antiproliferative activity of benzyl and phenethyl analogs of makaluvamines

Analogs of marine alkaloid, makaluvamine, bearing substituted benzyl and substituted phenethyl side chains have been synthesized and their antiproliferative activities have been evaluated. 4-Methyl, 4-chloro, and 4-fluoro substituted benzyl analogs possessed pronounced antiproliferative effects on the breast cancer cell line, MCF-7 at IC(50) values of 2.3 microM, 1.8 microM, and 2.8 microM, respectively. 4-Methyl, 4-chloro, and 3,4-methylenedioxy derivatives showed the best activity against MCF-7 among the phenethyl analogs with IC(50) values of 2.3 microM, 2.8 microM, and 2.4muM, respectively. In general, methoxy substitutions resulted in slight loss in activity in both benzyl and phenethyl series. Benzyl, 4-fluorobenzyl, 3,4-dimethoxyphenethyl, and 3,4-methylenedioxyphenethyl analogs were tested by NCI in their 60 cell lines in vitro human cancer cell screen. All four compounds showed excellent inhibition against several tested cancer cell lines. Benzyl and 4-fluorobenzyl analogs were relatively more active than 3,4-dimethoxy phenethyl and 3,4-methylenedioxy phenethyl analogs. In NCI assays, the best LogGI(50) values were shown by the fluorobenzyl analog against the renal cancer cell line RXF-393 (<-8.0M) and dimethoxy phenethyl analog against the CNS cancer cell line, SF-268 (<-8.0M). The best LogLC(50) value was shown by the fluorobenzyl analog against the breast cancer cell line MCF-7 (-6.01 M).     

Pathways of the rise and decay of the M photointermediate(s) of bacteriorhodopsin

The photocycle of bacteriorhodopsin (BR) was studied at alkaline pH with a gated multichannel analyzer, in order to understand the origins of kinetic complexities in the rise and decay of the M intermediate. The results indicate that the biphasic rise and decay kinetics are unrelated to a photoreaction of the N intermediate of the BR photocycle, proposed earlier by others [Kouyama et al. (1988) Biochemistry 27, 5855-5863]. Rather, under conditions where N did not accumulate in appreciable amounts (high pH, low salt concentration), they were accounted for by conventional kinetic schemes. These contained reversible interconversions, either M in equilibrium with N in one of two parallel photocycles or L in equilibrium with as well as M in equilibrium with N in a single photocycle. Monomeric BR also showed these kinetic complications. Conditions were then created where N accumulated in a photo steady state (high pH, high salt concentration, background illumination). The apparent increase in the proportion of the slow M decay component by the background illumination could be quantitatively accounted for with the single photocycle model, by the mixing of the relaxation of the background light induced photo steady state with the inherent kinetics of the photocycle. Postulating a new M intermediate which is produced by the photoreaction of N was neither necessary nor warranted by the data. The difference spectra suggested instead that absorption of light by N generates only one intermediate, observable between 100 ns and 1 ms, which absorbs near 610 nm. Thus, the photoreaction of N resembles in some respects that of BR containing 13-cis-retinal.     

Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.     

Effects of Asp-96----Asn, Asp-85----Asn, and Arg-82----Gln single-site substitutions on the photocycle of bacteriorhodopsin

Bacteriorhodopsin (BR) with the single-site substitutions Arg-82----Gln (R82Q), Asp-85----Asn (D85N), and Asp-96----Asn (D96N) is studied with time-resolved absorption spectroscopy in the time regime from nanoseconds to seconds. Time-resolved spectra are analyzed globally by using multiexponential fitting of the data at multiple wavelengths and times. The photocycle kinetics for BR purified from each mutant are determined for micellar solutions in two detergents, nonyl glucoside and CHAPSO, and are compared to results from studies on delipidated BR (d-BR) in the same detergents. D85N has a red-shifted ground-state absorption spectrum, and the formation of an M intermediate is not observed. R82Q undergoes a pH-dependent transition between a purple and a blue form with different pKa values in the two detergents. The blue form has a photocycle resembling that for D85N, while the purple form of R82Q forms an M intermediate that decays more rapidly than in d-BR. The purple form of R82Q does not light-adapt to the same extent as d-BR, and the spectral changes in the photocycle suggest that the light-adapted purple form of R82Q contains all-trans- and 13-cis-retinal in approximately equal proportions. These results are consistent with the suggestions of others for the roles of Arg-82 and Asp-85 in the photocycle of BR, but results for D96N suggest a more complex role for Asp-96 than previously suggested. In nonyl glucoside, the apparent decay of the M-intermediate is slower in D96N than in d-BR, and the M decay shows biphasic kinetics. However, the role of Asp-96 is not limited to the later steps of the photocycle. In D96N, the decay of the KL intermediate is accelerated, and the rise of the M intermediate has an additional slow phase not observed in the kinetics of d-BR. The results suggest that Asp-96 may play a role in regulating the structure of BR and how it changes during the photocycle.     

Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (lambda max = 500 nm) relative to that of native enzyme (lambda max = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, deadend complex formed by each analog is significantly blue-shifted (lambda max less than 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.     

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.