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1.
The photocycles of wild-type bacteriorhodopsin and its D96N form were investigated with a gated multichannel analyzer. Reconstruction of the spectra of the photointermediates from the measured time-resolved difference spectra allowed evaluation of the kinetics; the data at pH 7 in the presence of 100 mM NaCl were best fitted by the scheme K in eqiulibrium L in equilibrium M1----M2 in equilibrium N in equilibrium O----BR plus N----BR [Váró, G., & Lanyi, J. K. (1990) Biochemistry 29, 2241-2250]. The proposed two M states and the M1----M2 reaction were necessitated by anomalies in the kinetics of the decay of K and L. Additional support was provided by a 4-nm blue-shift in the maximum of M in Triton X-100 solubilized bacteriorhodopsin during the photocycle; the kinetics of the shift were consistent with the time course of the proposed M1----M2 transition. In the D96N mutant, the M state is stabilized, and the resulting equilibrium mixture for the intermediates could be evaluated with greater precision. The concentration ratio of L to M at the equilibrium was estimated to be no higher than 0.01. This requires the ratio of forward/reverse rates for the M1 to M2 conversion to be at least 200, i.e., a virtually irreversible reaction. Consistent with an earlier report, the data at lower pH and in the absence of NaCl are different and suggest the existence of a second L species; we propose that it is in equilibrium with M2. 相似文献
2.
Effects of the crystalline structure of purple membrane on the kinetics and energetics of the bacteriorhodopsin photocycle. 总被引:2,自引:0,他引:2
Time-resolved difference spectra were measured for Triton X-100 solubilized bacteriorhodopsin monomers between 100 ns and 100 ms after photoexcitation. The results are consistent with the general scheme K in equilibrium L in equilibrium M1 in equilibrium M2 in equilibrium N in equilibrium O----BR proposed previously for purple membranes [Váró, G., & Lanyi, J.K. (1990) Biochemistry 29, 2241-2250]. The rate constants which involve proton release or uptake, i.e., kLM1, kNO, and kON, were significantly higher in the monomeric protein than in purple membrane; the other steps were less affected. Analysis of the temperature dependencies of the rate constants between 5 and 30 degrees C yielded the enthalpies and entropies of activation for all steps except the two absent back-reactions. Comparison of these with data for purple membranes [Váró, G., & Lanyi, J.K. (1991) Biochemistry 30, 5016-5022] shows that the crystalline structure affects the energetics of the photocycle. In bacteriorhodopsin immobilized by the lattice of the purple membrane, the entropy changes leading to all transition states are more positive. Thus, the forward reactions proceed with less conformational hindrance. However, the thermal (enthalpic) barriers are higher. These effects are particularly pronounced for the M1----M2 and O----BR reactions. Large changes of the enthalpy and entropy levels of intermediates in the M2----BR reaction segment, but not in the K----M1 segment, upon solubilization of the protein are consistent with our earlier proposal that major protein conformational changes occur in the photocycle and they begin with the M1----M2 reaction. 相似文献
3.
The switch in the bacteriorhodopsin photocycle, which reorients access of the retinal Schiff base from the extracellular to the cytoplasmic side, was suggested to be an M1----M2 reaction (Váró and Lanyi. 1991. Biochemistry. 30:5008-5015, 5016-5022). Thus, in this light-driven proton pump it is the interconversion of proposed M substates that gives direction to the transport. We find that in monomeric, although not purple membrane-lattice immobilized, D115N bacteriorhodopsin, the absorption maximum of M changes during the photocycle: in the time domain between its rise and decay it shifts 15 nm to the blue relative to the spectrum at earlier times. This large shift strongly supports the existence of two M substates. Since D115 is located near the beta-ionone ring of the retinal, the result raises questions about the possible involvement of the retinal chain or protein residues as far away as 10 A from the Schiff base in the mechanism of the switching reaction. 相似文献
4.
Thermodynamics and energy coupling in the bacteriorhodopsin photocycle 总被引:18,自引:0,他引:18
Time-resolved absorption changes of photoexcited bacteriorhodopsin were measured with a gated multichannel analyzer between 100 ns and 100 ms at six temperatures between 5 and 30 degrees C. The energetics of the chromophore reaction cycle were analyzed on the basis of a model containing a single cycle and reversible reactions. The calculated thermodynamic parameters provide insights to general principles of the active transport. They indicate that in this light-driven proton pump the free energy is retained after absorption of the photon as the enthalpy of the pKa shift in the chromophore which allows deprotonation of the Schiff base. Part of the excess free energy is dissipated at the "switch" step where the reaction and transport cycles are coupled, and the rest at the chromophore recovery step. All other reactions take place near equilibrium. The "switch" step is the M1----M2 transition in the reaction cycle [Váró, G., & Lanyi, J. K. (1991) Biochemistry (preceeding paper in this issue)]. It provides for return of the chromophore pKa to its initial value so the Schiff base will become a proton acceptor, for reordering access of the Schiff base from one side of the membrane to the other, and for unidirectionality of the proton transfer. Conformational energy of the protein, acquired during the "switch" step, drives the completion of the photocycle. 相似文献
5.
Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization. 相似文献
6.
The changes in the photocycle of the wild type and several mutant bacteriorhodopsin (D96N, E204Q, and D212N) were studied on dried samples, at relative humidities of 100% and 50%. Samples were prepared from suspensions at pH approximately 5 and at pH approximately 9. Intermediate M with unprotonated Schiff base was observed at the lower humidity, even in the case where the photocycle in suspension did not contain this intermediate (mutant D212N, high pH). The photocycle of the dried sample stopped at intermediate M1 in the extracellular conformation; conformation change, switching the accessibility of the Schiff base to the cytoplasmic side, and proton transport did not occur. The photocycle decayed slowly by dissipating the absorbed energy of the photon, and the protein returned to its initial bacteriorhodopsin state, through several M1-like substates. These substates presumably reflect different paths of the proton back to the Schiff base, as a consequence of the bacteriorhodopsin adopting different conformations by stiffening on dehydration. All intermediates requiring conformational change were hindered in the dried form. The concentration of intermediate L, which appears after isomerization of the retinal from all-trans to 13-cis, during local relaxation of the protein, was unusually low in dried samples. The lack of intermediates N and O demonstrated that the M state did not undergo a change from the extracellular to the cytoplasmic conformation (M1 to M2 transition), as already indicated by Fourier transform infrared spectroscopy, quasielastic incoherent neutron scattering, and electric signal measurements described in the literature. 相似文献
7.
The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of their interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Váró, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BRhv-->K<==>L<==>M1-->M2--> BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3 x 3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25 degrees C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein. 相似文献
8.
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. 相似文献
9.
Pharaonis halorhodopsin (phR), the light-driven chloride ion pump from Natronobacterium pharaonis with C-terminal histidine tag, was expressed in Escherichia coli cells. The protein was solubilized with 0.1% n-dodecyl beta-D-maltopyranoside and purified with a nickel column. Removal of Cl- from the medium yields blue phR (phR(blue)) that has lost Cl- near the chromophore. Addition of Cl- converts phR(blue) to a red-shifted Cl--bound form (phR(Cl)). Circular dichroic spectra of phR(blue) and phR(Cl) exhibited a bilobe in the visual region, indicating specific oligomerization of the phR monomers. The order of anion concentration which induced a shift from phR(blue) to phR(X) was Br- < Cl- < NO3- < N3-, which was the same as in the case of phR purified from N. pharaonis membranes. Chloride binding kinetics was measured by time-resolved absorption changes with stopped-flow rapid mixing. Rates of Cl- binding consisted of fast and slow components, and the amplitude of the fast component was about 90% of the total changes. The rate constant of the fast component at 100 mM NaCl at 25 degrees C was 260 s(-1) with an apparent activation energy of 35 kJ/mol. These values are in good agreement with the process of Cl- uptake in the photocycle (O --> hR' reaction) reported previously [Váró et al. (1995) Biochemistry 34, 14500-14507]. In addition, the Cl- concentration dependence on both rates was similar to each other. These observations suggest that the O-intermediate is similar to phR(blue) and that Cl- uptake during the photocycle may be ruled by a passive process. 相似文献
10.
11.
G A Perkins E Liu F Burkard E A Berry R M Glaeser 《Journal of structural biology》1992,109(2):142-151
A combination of visible and Fourier transform infrared (FTIR) spectroscopies is used to characterize the formation of the M1 and M2 substates of the bacteriorhodopsin photocycle in glucose-embedded, hydrated thin films. Difference FTIR bands in the amide I region verify the previously reported existence of a significant peptide backbone conformational change in the transition from M1 to M2. The visible absorption spectra demonstrate that contamination of the M-intermediate samples by L, N, or other non-M species should contribute negligibly to the observed changes in the amide I region, and this conclusion is supported by comparison of specific carboxyl group peaks with corresponding bands in published L and N FTIR difference spectra. Based upon spectroscopic results, an extension of the C-T Model (Fodor, S., Ames, J., Gebhard, R., van den Berg, E., Stoeckenius, W., Lugtenberg, J., and Mathies, R. (1988) Biochemistry 27, 7097-7101) is presented. The results of this work suggest that protein structural changes should be clearly visible in M-bR, difference Fourier density maps and that these structural changes may in turn elucidate how bacteriorhodopsin actively pumps ions across the purple membrane of Halobacterium halobium. 相似文献
12.
R Needleman M Chang B Ni G Váró J Fornés S H White J K Lanyi 《The Journal of biological chemistry》1991,266(18):11478-11484
The gene coding for bacteriorhodopsin was modified in vitro to replace Asp212 with asparagine and expressed in Halobacterium halobium. X-ray diffraction measurements showed that the major lattice dimension of purple membrane containing the mutated bacteriorhodopsin was the same as wild type. At pH greater than 7, the Asp212----Asn chromophore was blue (absorption maximum at 585 nm) and exhibited a photocycle containing only the intermediates K and L, i.e. a reaction sequence very similar to that of wild-type bacteriorhodopsin at pH less than 3 and the blue form of the Asp85----Glu protein at pH less than 9. Since in the latter cases these effects are attributed to protonation of residue 85, it now appears that removal of the carboxylate of Asp212 has similar consequences as removing the carboxylate of Asp85. However, an important difference is that only Asp85 affects the pKa of the Schiff base. At pH less than 7, the Asp212----Asn protein was purple (absorption maximum at 569 nm) but photoexcitation produced only 15% of the normal amount of M and the transport activity was partial. The reactions of the blue and purple forms after photoexcitation are both quantitatively accounted for by a proposed scheme, K in equilibrium with L1 in equilibrium with L2----BR, but with the addition of an L1 in equilibrium with M reaction with unfavorable pKa for Schiff base deprotonation in the purple form. The latter hinders the transient accumulation of M, and the consequent branching at L1 allows only partial proton transport activity. The results are consistent with the existence of a complex counterion for the Schiff base proposed earlier (De Groot, H. J. M., Harbison, G. S., Herzfeld, J., and Griffin, R. G. (1989) Biochemistry 28, 3346-3353) and suggest that Asp85, Asp212, and at least one other protonable residue participate in it. 相似文献
13.
In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures. 相似文献
14.
Below 195 K, the bacteriorhodopsin photocycle could not be adequately described with exponential kinetics [Dioumaev, A. K., and Lanyi, J. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 9621-9626] but required distributed kinetics, previously found in hemoglobin and myoglobin at temperatures below the vitrification point of the surrounding solvent. The aim of this study is to determine which factors cause the switch from this low-temperature regime to the conventional kinetics observed at ambient temperature. The photocycle was monitored by time-resolved FTIR between 180 and 280 K, using the D96N mutant. Depending on the temperature, decay and temporal redistribution of two or three intermediates (L, M, and N) were observed. Above approximately 245 K, an abrupt change in the kinetic behavior of the photocycle takes place. It does not affect the intermediates present but greatly accelerates their decay. Below approximately 240 K, a kinetic pattern with partial decay that cannot be explained by conventional kinetics, but suggesting distributed kinetics, was dominant, while above approximately 250 K, there were no significant deviations from exponential behavior. The approximately 245 K critical point is >/=10 K below the freezing point of interbilayer water, and we were unable to correlate it with any FTIR-detectable transition of the lipids. Therefore, we attribute the change from distributed to conventional kinetics to a thermodynamic phase transition in the protein. Most probably, it is related to the freezing and thawing of internal fluctuations of the protein, known as the dynamic phase transition, although in bacteriorhodopsin the latter is usually believed to take place at least 15 K below the observed critical temperature of approximately 245 K. 相似文献
15.
Transient photovoltages in purple membrane multilayers. Charge displacement in bacteriorhodopsin and its photointermediates 总被引:2,自引:0,他引:2
The photovoltaic properties of bacteriorhodopsin molecules and their photochemical intermediates have been investigated in an experimental cell consisting of multilayered films of highly oriented, dry fragments of purple membrane and lipid sandwiched between two metal (Pd) electrodes. The electrical time constant of these sandwich cells containing between 5 and 30 layers is less than 10(-5) S. Bright illumination of these cells with actinic flashes of approximately 1 ms duration generates transient photovoltages. These photovoltages, which make the extracellular surface of purple membrane positive with respect to the intracellular surface, follow the time course of the flash with no detectable latency. The amplitude of the photovoltages increases linearly with light intensity and their action spectrum matches the absorption spectrum of the light-adapted state of bacteriorhodopsin, BR570. In these dry multilayer cells, the slow photointermediates of bacteriorhodopsin, M412, N520 and O640 are long lived. Illumination of the sandwich cells with long duration (200 ms) pulses of light results, therefore, in the formation of photomixtures containing all these slow photointermediates. Flash illumination of the sandwich cells immediately following the conditioning pulse produces photovoltages whose action spectra match the absorption spectra of the M412 and N520 photointermediates. The M412 photovoltages, like the BR570 photovoltages, follow the time course of the actinic flash with no detectable latency and increase in amplitude linearly with light intensity. But, unlike the BR570 photovoltage, the M412, N520 and O640 photovoltages make the extracellular surface of purple membrane negative with respect to the intracellular surface. Through the of their specific photovoltaic signals, M412 and N520 are shown to be kinetically distinct photointermediates of bacteriorhodopsin. Detection of fast photovoltages with these characteristics in the absence of any ionic solution, and in parallel with spectrophotometric changes, suggest that they arise from charge displacements in the bacteriorhodopsin molecules and their photointermediates as they undergo photochemical conversion in response to the absorption of photons. 相似文献
16.
Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates 总被引:6,自引:0,他引:6
The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups. 相似文献
17.
The absorption spectrum of light-adapted purple membrane in 3 M KCl is dependent on temperature even in the room temperature region. Temperature-induced difference spectra at various pH values suggested that the trans isomer of bacteriorhodopsin, bR570, is in thermal and/or photodynamic equilibrium with several different conformers. The major second conformer occurring at neutral pH had the same spectroscopic properties as the 13-cis isomer, and its content at 35 degrees C was estimated to be more than 20%. Heterogeneity in the protein conformation became more significant above pH8, where temperature-induced difference spectra exhibited a negative peak at 580 nm and a positive peak at 296 nm. This absorption change is very similar to that observed upon the formation of the N intermediate, suggesting that an N-like conformer occurs at high pH and temperature. A significant temperature dependence was also seen in the M decay kinetics at high pH, which were described by two decay components; i.e., the fast decaying M (Mf) was predominant at low temperature, but the amplitude of the slow component (M(s)) increased with increasing temperature. It is suggested that M(s) is generated upon excitation of the N-like conformer, in which the residue (Asp-96) usually acting as a proton donor to the Schiff base is deprotonated. The N-like conformer could be N itself, because M(s) was enhanced when N was accumulated by background light. A strong correlation between the amplitude of M(s) and the concentration of N was also revealed by the accumulation kinetics of Mf, M(s), and N after the onset of continuous actinic light.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump. 相似文献
19.
Testing BR photocycle kinetics. 总被引:4,自引:1,他引:3
An improved K absorption spectrum in the visible is obtained from previous photocycle data for the D96N mutant of bacteriorhodopsin, and the previously obtained M absorption spectrum in the visible and the fraction cycling are confirmed at 25 degrees C. Data at lower temperatures are consistent with negligible temperature dependence in the spectra from 5 degrees C to 25 degrees C. Detailed analysis strongly indicates that there are two intermediates in addition to the first intermediate K and the last intermediate M. Assuming two of the intermediates have the same spectrum and using the L spectrum obtained previously, the best kinetic model with four intermediates that fits the time course of the intermediates is rather unusual, with two L's on a cul-de-sac. However, a previously proposed, more conventional model with five intermediates, including two L's with the same spectra and two M's with the same spectra, also fits the time course of the intermediates nearly as well. A new criterion that tests an individual proposed spectrum against data is also proposed. 相似文献
20.
Analogies between halorhodopsin and bacteriorhodopsin 总被引:6,自引:0,他引:6
Váró G 《Biochimica et biophysica acta》2000,1460(1):220-229
The light-activated proton-pumping bacteriorhodopsin and chloride ion-pumping halorhodopsin are compared. They belong to the family of retinal proteins, with 25% amino acid sequence homology. Both proteins have seven alpha helices across the membrane, surrounding the retinal binding pocket. Photoexcitation of all-trans retinal leads to ion transporting photocycles, which exhibit great similarities in the two proteins, despite the differences in the ion transported. The spectra of the K, L, N and O intermediates, calculated using time-resolved spectroscopic measurements, are very similar in both proteins. The absorption kinetic measurements reveal that the chloride ion transporting photocycle of halorhodopsin does not have intermediate M characteristic for deprotonated Schiff base, and intermediate L dominates the process. Energetically the photocycle of bacteriorhodopsin is driven mostly by the decrease of the entropic energy, while the photocycle of halorhodopsin is enthalpy-driven. The ion transporting steps were characterized by the electrogenicity of the intermediates, calculated from the photoinduced transient electric signal measurements. The function of both proteins could be described with the 'local access' model developed for bacteriorhodopsin. In the framework of this model it is easy to understand how bacteriorhodopsin can be converted into a chloride pump, and halorhodopsin into a proton pump, by changing the ion specificity with added ions or site-directed mutagenesis. 相似文献