Wild-type bR Research Articles

Photoproducts of bacteriorhodopsin mutants: a molecular dynamics study

Molecular dynamics simulations of wild-type bacteriorhodopsin (bR) and of its D85N, D85T, D212N, and Y57F mutants have been carried out to investigate possible differences in the photoproducts of these proteins. For each mutant, a series of 50 molecular dynamics simulations of the photoisomerization and subsequent relaxation process were completed. The photoproducts can be classified into four distinct classes: 1) 13-cis retinal, with the retinal N-H+ bond oriented toward Asp-96; 2) 13-cis retinal, with the N-H+ oriented toward Asp-85 and hydrogen-bonded to a water molecule; 3) 13,14-di-cis retinal; 4) all-trans retinal. Simulations of wild-type bR and of its Y57F mutant resulted mainly in class 1 and class 2 products; simulations of D85N, D85T, and D212N mutants resulted almost entirely in class 1 products. The results support the suggestion that only class 2 products initiate a functional pump cycle. The formation of class 1 products for the D85N, D85T, and D212N mutants can explain the reversal of proton pumping under illumination by blue and yellow light.

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.

NONLINEAR OPTICAL STUDIES OF BACTERIORHODOPSIN

We report interesting results on nonlinear optics at low powers in bacteriorhodopsin films with applications in all-optical switching and modulation. Chemically stabilized films of bacteriorhodopsin in a polymer matrix for which the lifetime of the excited M state is 3 to 4 orders of magnitude longer than that of water solutions of wild-type bR were used in these experiments. Due to the sensitivity of the films, very small powers of order microwatts are required for optical phase conjugation. The influence of the fast photochemical M to B transition induced by blue light on the saturation intensity, phase conjugate intensity and switching time was established. We also report our measurements of the intensity dependence of the self-focusing and self-defocusing properties of wild-type bR in water solution using the Z-scan technique with low power cw lasers at two wavelengths on either side of the absorption band. Our measurements indicate that the sign of the nonlinearity depends on the wavelength and the magnitude depends on the fluence of the incident laser beam. The observed self-focusing and defocusing is not due to the intrinsic electronic nonlinearity. The observations can be explained in terms of the Kramers-Kronig dispersion relation that relates the real and imaginary parts of the complex index of refraction.

Nanosecond optical excitation dynamics of long M-state lifetime bacteriorhodopsin films

Dynamic parameters relevant to the performance of bacteriorhodopsin (bR) as an optical cache memory are reported. The thermal relaxation of the M-state photocycle-intermediate has been a barrier to the exploitation of wild-type bR in optical memory devices. Extension of the room-temperature M-state lifetime from 10 ms to ∼10 s is described. Writing to the long-lifetime material is achieved for nanosecond pulses of less than 1 nJ μm −2 fluence. Reading, at a contrast-ratio of 1.3:1, is achievable following a 5 ms delay. Erasure is achieved on a 50 μs timescale for pulse fluences similar to those used for the writing stage. Following continuous-wave (cw) excitation, contrast ratios of 200:1 are obtained, with significant photochromic changes remaining after many seconds.

Cooperative phenomena in the photocycle of D96N mutant bacteriorhodopsin

The M intermediate decay in the photocycle of D96N mutant bacteriorhodopsin does not depend on the light intensity of the exciting flash. Cooperative phenomena in the photocycle are revealed after addition of azide causing acceleration of the M decay and making it kinetically well separated from the N decay. Increase in the light intensity induces slight deceleration of the M decay and significant acceleration of the N decay. The data obtained directly confirm our recent model [ Komrakov and Kaulen (1995) Biophys. Chem. 56, 113–119], according to which appearance of the M slow intermediate in the photocycle of the wild type bR at high light intensity is due to destabilization of the N intermediate leading to the acceleration of the N→M and N→bR reactions.

Asp 46 can substitute for Asp 96 as the Schiff base proton donor in bacteriorhodopsin

Bacteriorhodopsin functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium. A variety of studies have established that a proton is transferred over an approximately 10 A distance from Asp 96 to the retinylidene Schiff base during the M --> N transition of the bR photocycle. In order to further explore the mechanism of this Schiff base reprotonation, we compared the properties of the double mutant Thr 46 --> Asp/Asp 96 --> Asn (T46D/D96N), the single mutants Asp 96 --> Asn (D96N) and Thr 46 --> Asp (T46D), and wild-type bR. In contrast to D96N, which exhibits a very slow M decay, T46D/D96N has an M decay close to that of wild-type bR. FTIR difference spectroscopy detects bands in the carboxyl and carboxylate stretch region of T46D/D96N consistent with the deprotonation of Asp 46 during the M --> N transition. In addition, bands associated with structural changes of Asn 96 in the mutant D96N are absent in T46D/D96N. Resonance Raman spectroscopy provides evidence that both T46D/D96N and T46D have a long-lived N-like species in their photocycles. These data demonstrate that Asp 46 can substitute for Asp 96 as the proton donor group in the reprotonation pathway of the Schiff base during the M --> N transition. However, N decay is delayed in comparison to wild-type bR. This may be due to a partial block in the proton pathway leading from the cytoplasmic medium to Asp 46.

Bacteriorhodopsin can function without a covalent linkage between retinal and protein

Light energy is transferred from retinal to the protein in bacteriorhodopsin after absorption of a photon resulting in changes of protein conformation. To examine whether the covalent bond, formed by the carbonyl group of retinal and the epsilon-amino group of lysine 216, is essential for this process, a mutant with lysine 216 replaced by alanine was expressed in Halobacterium salinarium L33 (BO-, retinal+). Reconstitution of the chromoprotein with varying retinylidene-n-alkylamines was possible in isolated membranes as well as in whole cells. When the protein in membranes with retinylidene Schiff bases of n-alkylamines of different lengths was reconstituted, the most stable chromoprotein was formed with retinylideneethylamine. The absorbance maximum was at 475 nm in alkaline solution and 620 nm in acidic solution. At neutral pH values both species equilibrate with a third one absorbing maximally at 568 nm. Reconstitution of whole cells with retinylideneethylamine led to a specific proton pump activity of 30 mol of protons per mol of BR per minute. This value indicates a lower limit of transport; no light saturation could be reached in these measurements in contrast to wild-type BR where transport activities of 162 mol of protons per mol of BR per minute under identical conditions can be achieved. Action spectra from flash photolysis experiments revealed that only the 568-nm form led to a M-intermediate with a half-time of decay of 17 ms. In summary, it could be shown that the covalent linkage between retinal and the protein is basically not required for the function of bacteriorhodopsin as a light-driven proton pump.

X-ray diffraction of a cysteine-containing bacteriorhodopsin mutant and its mercury derivative. Localization of an amino acid residue in the loop of an integral membrane protein.

We have used heavy-atom labeling and X-ray diffraction to localize a single amino acid in the integral membrane protein bacteriorhodopsin (bR). To provide a labeling site, we used the bR mutant, A103C, which contains a unique cysteine residue in the short loop between transmembrane alpha-helices C and D. The mutant protein was expressed in and purified from Halobacterium halobium, where it forms a two-dimensional crystalline lattice. In the lattice form, the protein reacted with the sulfhydryl-specific reagent p-chloromercuribenzoate (p-CMB) in a 1:0.9 stoichiometry to yield the p-mercuribenzoate derivative (A103C-MB). The functional properties of A103C and A103C-MB, including the visible absorption spectrum, light-dark adaptation, photocycle, and proton release kinetics, were similar to those of wild-type bR. X-ray diffraction experiments demonstrated that A103C and A103C-MB membranes have the same hexagonal protein lattice as wild-type purple membrane. Thus, neither the cysteine substitution nor mercury labeling detectably affected bR structure or function. By using Fourier difference methods, the in-plane position of the mercuribenzoate label was calculated from intensity differences in the X-ray diffraction patterns of A103C and A103C-MB. This analysis revealed a well-defined mercury peak located between alpha-helices C and D. The approach reported here offers promise for refining the bR structural model, for monitoring conformational changes in bR photointermediates, and for studying the structure of other proteins in two-dimensional crystals.

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum

The structure of mutant bacteriorhodopsin (bR), D85N, was examined by CD and X-ray diffraction at pH 7. The absorption maximum of D85N at pH 7 is located at 605 nm, which is similar to the acid-blue form of wild-type bR. D85N shows a monophasic CD band, the maximum of which is at 575 nm, although the crystalline arrangement and the trimeric structure is maintained. The acid-blue form of wild-type bR shows a biphasic CD despite the similarity in absorption spectra.

Two-dimensional crystallization of Escherichia coli-expressed bacteriorhodopsin and its D96N variant: high resolution structural studies in projection

Highly ordered two-dimensional (2-D) crystals of Escherichia coli-expressed bacteriorhodopsin analog (e-bR) and its D96N variant (e-D96N) reconstituted in Halobacterium halobium lipids have been obtained by starting with the opsin protein purified in the denaturing detergent sodium dodecyl sulfate. These crystals embedded in glucose show electron diffraction in projection to better than 3.0 A at room temperature. This is the first instance that expressed bR or a variant has been crystallized in 2-D arrays showing such high order. The crystal lattice is homologous to that in wild-type bR (w-bR) in purple membranes (PM) and permit high resolution analyses of the structure of the functionally impaired D96N variant. The e-bR crystal is isomorphous to that in PM with an overall averaged fractional change of 12.7% (26-3.6-A resolution) in the projection structure factors. The projection difference Fourier map e-bR-PM at 3.6-A resolution indicates small conformational changes equivalent to movement of approximately < 7 C-atoms distributed within and in the neighborhood of the protein envelope. This result shows that relative to w-bR there are no global structural rearrangements in e-bR at this 3.6 A resolution level. The e-D96N crystal is isomorphous to the e-bR crystal with a smaller (9.2%) overall averaged fractional change in the structure factors. The significant structural differences between e-D96N and e-bR are concentrated at high resolution (5-3.6 A); however, these changes are small as quantified from the 3.6 A resolution e-D96N-e-bR Fourier difference map. The difference map showed no statistically significant peaks or valleys within 5 A in projection from the site of D96 substitution on helix C. Elsewhere within the protein envelope the integrated measure of peaks or valleys was < approximately 3 C-atom equivalents. Thus, our results show that for the isosteric substitution of Asp96 by Asn, the molecular conformation of bR in its ground state is essentially unaltered. Therefore, the known effect of D96N on the slowed M412 decay is not due to ground-state structural perturbations.

Hydrogen bonding interactions with the Schiff base of bacteriorhodopsin. Resonance Raman spectroscopy of the mutants D85N and D85A.

The bacteriorhodopsin (bR) mutants Asp-85-->Asn (D85N) and Asp-85-->Ala (D85A) have a red-shifted chromophore absorption and exhibit no proton pumping (Otto, H., Marti, T., Holz, M., Mogi, T., Stern, L., Engel, F., Khorana, H. G., and Heyn, M. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1018-1022) consistent with the hypothesis that Asp-85 functions as a counterion and proton acceptor for the retinal Schiff base (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520). Resonance Raman spectroscopy reveals that these mutants contain a mixture of all-trans and 13-cis/C = N syn chromophores, similar to dark-adapted purple membrane and acid-induced or deionized blue membrane. At high NaCl concentrations, both mutants adopt a predominantly all-trans chromophore structure similar to acid purple membrane. A comparison of the Schiff base C = NH+ stretch frequency (vC = N) and deuterium isotope shift for D85N, D85A as well as various forms of bR, including light-adapted bR, blue membrane, and acid purple membrane, provides information about hydrogen bonding interactions to the Schiff base. D85N has as strong a hydrogen bond as light-adapted bR despite the loss of the negative charge at residue 85. In contrast, D85A has a weaker hydrogen bond. These results can be explained if a direct interaction exists between the Schiff base and Asn-85 in D85N and between the Schiff base and a substituted water molecule in D85A. Many of the properties of wild type bR, D85N, D85A, blue membrane, and acid purple membrane can be explained on the basis of changes in the local hydrogen bonding near the Schiff base.

Effects of mutagenetic substitution of prolines on the rate of deprotonation and reprotonation of the Schiff base during the photocycle of bacteriorhodopsin.

Membrane-buried proline residues are found in many transport proteins. To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions of Pro-50, Pro-91 and Pro-186 on the deprotonation and reprotonation kinetics of the Schiff base (SB) were determined by flash photolysis. The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli). The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant. These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate. Our results indicate that Pro-50 and Pro-91, as well as Pro-186, are important for the proton-pumping function of bR.

FTIR difference spectroscopy of the bacteriorhodopsin mutant Tyr-185 .fwdarw. Phe: Detection of a stable O-like species and characterization of its photocycle at low temperature

Fourier transform infrared difference spectroscopy has been used to study the photocycle of the mutant Tyr-185-->Phe expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. We find several changes in the low-temperature bR-->K, bR-->L, and bR-->M FTIR difference spectra of Y185F relative to wild-type bR which are not directly related to the absorption bands associated with Tyr-185. We show that these features arise from the photoreaction of a stable red-shifted species (OY185F) with a vibrational spectrum similar to the O intermediate. By using photoselection and FTIR spectroscopy, we have been able to characterize the photoproducts of this OY185F species. A K-like photoproduct is formed at 80 K which has a 13-cis structure. However, it differs from K630, exhibiting an intense band at 990 cm-1 most likely due to a hydrogen-out-of-plane vibrational mode of the chromophore. At 170 and 250 K, photoexcitation of OY185F produces an intermediate with vibrational features similar to the N intermediate in the wild-type bR photocycle. However, no evidence for an M-like intermediate is found. Although Asp-96 undergoes a change in its environment/protonation state during the OY185F photocycle, no protonation changes involving Asp-85 and Asp-212 were detected. These results provide strong evidence that light adaptation of Y185F produces two species similar to bR570 and the O intermediate. Differences in their respective photocycles can be explained on the basis of differences in the protonation states of the residues Asp-85 and Asp-212 which are ionized in bR570 and undergo net protonation upon OY185F formation.

Fourier transform Raman spectroscopy of the bacteriorhodopsin mutant Tyr-185 .fwdarw. Phe: Formation of a stable O-like species during light adaptation and detection of its transient N-like photoproduct

Near-infrared FT-Raman spectroscopy can be used to measure the vibrations of the bacteriorhodopsin (bR) chromophore without the disadvantage of conventional visible resonance Raman spectroscopy, where the visible excitation drives the bR photoreactions. We utilized this technique to investigate the light-dark adaptation of bacteriorhodopsin and the mutant Tyr-185-->Phe (Y185F) at room temperature in solution. Compared to wild-type bR, both the FT-Raman and resonance Raman spectra of the light-adapted Y185F displayed new features characteristic of the vibrations of the O intermediate. Light adaptation of Y185F was found to involve a 13-cis, C=N syn-->all-trans isomerization of the retinal chromophore which produces a species similar to bR570 and a second O-like species. Dark adaptation, which was much slower in Y185F compared to wild-type bR, involved a parallel decay of the bR570 and O-like species and resulted in a decreased all-trans:13-cis ratio compared to wild type. Further evidence for the existence of an O-like species in Y185F comes from pump-probe Raman difference spectroscopy, where a red pump beam is found to produce a species very similar to the N intermediate in the photocycle. This species is shown by stroboscopic Raman measurements to exist transiently even at high pH. We postulate that when the Y185F chromophore has an all-trans structure the effective pKa of Asp-85 and Asp-212 is elevated in Y185F due to the disruption of the Asp-212/Tyr-185 hydrogen bond, thereby accounting for the increased protonation of these residues in the O-like species.

Static and time-resolved absorption spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: evidence for an equilibrium between bR570 and an O-like species.

The light-dark adaptation, photocycle kinetics, and acid-induced blue formation of the bacteriorhodopsin (bR) mutant Tyr-185-->Phe (Y185F) expressed in Halobacterium halobium have been investigated by both static and time-resolved visible absorption spectroscopy. Evidence is presented that a pH-dependent equilibrium exists between a bR570-like form (bRY185F570) and a red-shifted species in the light-adapted form of Y185F. In two related papers, we show that this species has vibrational features similar to the O intermediate. Key findings are that light adaptation causes formation of a purple species similar to bR570 and a second long-lived red-shifted species with a lambda max near 630 nm, well above the pH for the acid-induced blue transition. The concentration of the red-shifted species is pH- and salt-dependent, decreasing reversibly at high pH and high ionic strength. The dark-adapted state of Y185F also contains a small amount of the red-shifted species which is reversibly titratable. Dark adaptation is much slower than wild-type bR and causes a parallel decay of light-adapted bR and the red-shifted species. Time-resolved visible absorption spectroscopy reveals that the purple and the red-shifted species undergo separate photocycles. The purple species exhibits a relatively normal photocycle except for an increased rate of M formation kinetics. The red-shifted species has a photocycle involving a red-shifted K intermediate and a second longer lived intermediate possibly similar to N. The apparent absence of an O intermediate in the late photocycle of Y185F is attributed to cancellation by depletion bands due to the photoreacting red-shifted species.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of genetic replacements of charged and H-bonding residues in the retinal pocket on Ca2+ binding to deionized bacteriorhodopsin.

Metal cations are known to be required for proton pumping by bacteriorhodopsin (bR). Previous studies found that bR has two high-affinity and four to six low-affinity Ca(2+)-binding sites. In our efforts to find the location of these Ca2+ sites, the effects of replacing charged (Asp-85, Asp-212, and Arg-82) and H-bonding (Tyr-185) residues in the retinal pocket on the color control and binding affinity of Ca2+ ions in Ca(2+)-regenerated bR were examined. The important results are as follows: (i) The removal of Ca2+ from recombinant bR in which charged residues were replaced by neutral ones shifted the retinal absorption to the blue, opposite to that observed in wild-type bR or in recombinant bR in which the H-bonding residue, Tyr-185, was replaced by a non-H-bonding amino acid (Phe). (ii) Similar to the observation in wild-type bR, the binding of Ca2+ to the second site gave the observed color change in the recombinant bR samples in which charged residues were replaced by neutral ones. (iii) The residue replacements had no effect on the affinity constants of the four to six weakly bound Ca2+. (iv) The two high-affinity sites exhibited reduced affinity with substitutions; while the extent of the reduction depended on the specific substitution, each site was reduced by the same factor for each of the charged residue substitutions but by different factors for the mutant where Tyr-185 was replaced with Phe(Y185F). The above results suggest that the two Ca2+ ions in the two high-affinity sites are within interaction distance with one another and with the charged residues in the retinal pocket. The results further suggest that, while the interaction between Tyr-185 and the high-affinity Ca2+ ions is relatively short range and specific (with more coupling to the Ca2+ ion in the second affinity site), between the charged residues and Ca2+ ions it seems to be of the electrostatic (e.g., ion-ion) long range, nonspecific type. Although neither Asp-85, Asp-212, nor Arg-82 is individually directly involved in the binding of Ca2+ in these two sites, they might all participate in it. Together with the protonated Schiff base, the charged residues along with Tyr-185 and one or two Ca2+ ions (and probably a few water molecules) seem to form an electrostatically coupled system that is part of a cavity that controls the color and function of bR.

Bacteriorhodopsin D85N: Three spectroscopic species in equilibrium

Ground-state absorbance measurements show that BR from Halobacterium halobium containing asparagine at residue 85 (D85N) exists as three distinct chromophoric states in equilibrium. In the pH range 6-12 the absorbance spectra of the three states are demonstrated to be similar to flash-induced spectral intermediates which comprise the latter portion of the wild-type BR photocycle. One of the states absorbs maximally at 405 nm, has a deprotonated Schiff base, and contains predominantly the 13-cis form of retinal, identifying it as a close homologue of the M intermediate in the BR photocycle. The other species possess absorbance maxima with correspondence to those of the wild-type N (570 nm) and O (615 nm) photointermediates. The retinal composition of the O-like form was found to be dominated by all-trans isomer. The pH dependence of the concentrations of the equilibrium species corresponds closely with the pH dependence of the M, N, and O photointermediates. These data support kinetic models which emphasize the role of back-reactions during the photocycle of bacteriorhodopsin. Energetic and spectral characterization of the D85N ground-state equilibrium supports its use as a model for elucidating molecular transitions comprising the latter portion of the BR photocycle.

Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation; Asp-85 and Asp-212 deprotonate during O decay.

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

Interrelations of M-intermediates in bacteriorhodopsin photocycle

The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by the flash-photolysis technique. The M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, τ 1 = 65 and τ 2 = 250 μs) for the wild-type bR and three exponents (τ 1 = 55 μs, τ 2 = 220 μs and τ 3 = 1 ms) for the D96N mutant of bR. A component with τ = 1 ms was found to be present in the photocycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu 3+ ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the 1-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp 96→Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild-type bR and D96N mutant and matched the M→M′ conformational transition. A model for M rise in the bR photocycle is proposed.

Spectral relationship of light-induced refractive index and absorption changes in bacteriorhodopsin films containing wildtype BRWT and the variant BRD96N

Bacteriorhodopsin films containing the variant BR D96N , which differs from the wildtype BR WT by a single amino acid exchange, Asp 96→Asn, show significantly higher holographic diffraction efficiencies(η) than BR WT films. The light-induced changes of both the absorption a(λ,I) and the refractive index n(λ,I), which influence the diffraction efficiency η(λ,I), have been investigated, and their spectral dependence has been measured in order to analyze both parameters separately