Molecular dynamics study of the M412 intermediate of bacteriorhodopsin.

Molecular dynamics simulations have been carried out to study the M412 intermediate of bacteriorhodopsin's (bR) photocycle. The simulations start from two simulated structures for the L550 intermediate of the photocycle, one involving a 13-cis retinal with strong torsions, the other a 13,14-dicis retinal, from which the M412 intermediate is initiated through proton transfer to Asp-85. The simulations are based on a refined structure of bR568 obtained through all-atom molecular dynamics simulations and placement of 16 waters inside the protein. The structures of the L550 intermediates were obtained through simulated photoisomerization and subsequent molecular dynamics, and simulated annealing. Our simulations reveal that the M412 intermediate actually comprises a series of conformations involving 1) a motion of retinal; 2) protein conformational changes; and 3) diffusion and reconfiguration of water in the space between the retinal Schiff base nitrogen and the Asp-96 side group. (1) turns the retinal Schiff base nitrogen from an early orientation toward Asp-85 to a late orientation toward Asp-96; (2) disconnects the hydrogen bond network between retinal and Asp-85 and tilts the helix F of bR, enlarging bR's cytoplasmic channel; (3) adds two water molecules to the three water molecules existing in the cytoplasmic channel at the bR568 stage and forms a proton conduction pathway. The conformational change (2) of the protein involves a 60 degrees bent of the cytoplasmic side of helix F and is induced through a break of a hydrogen bond between Tyr-185 and a water-side group complex in the counterion region.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that ASP-96 deprotonates during the M----N transition

The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96----Ala and Asp-96----Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO- vibration. The Asp-96----Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L----M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N----O or O----bR transition.     

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.     

Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release.

Titration of Asp-85, the proton acceptor and part of the counterion in bacteriorhodopsin, over a wide pH range (2-11) leads us to the following conclusions: 1) Asp-85 has a complex titration curve with two values of pKa; in addition to a main transition with pKa = 2.6 it shows a second inflection point at high pH (pKa = 9.7 in 150-mM KCl). This complex titration behavior of Asp-85 is explained by interaction of Asp-85 with an ionizable residue X'. As follows from the fit of the titration curve of Asp-85, deprotonation of X' increases the proton affinity of Asp-85 by shifting its pKa from 2.6 to 7.5. Conversely, protonation of Asp-85 decreases the pKa of X' by 4.9 units, from 9.7 to 4.8. The interaction between Asp-85 and X' has important implications for the mechanism of proton transfer. In the photocycle after the formation of M intermediate (and protonation of Asp-85) the group X' should release a proton. This deprotonated state of X' would stabilize the protonated state of Asp-85.2) Thermal isomerization of the chromophore (dark adaptation) occurs on transient protonation of Asp-85 and formation of the blue membrane. The latter conclusion is based on the observation that the rate constant of dark adaptation is directly proportional to the fraction of blue membrane (in which Asp-85 is protonated) between pH 2 and 11. The rate constant of isomerization is at least 10(4) times faster in the blue membrane than in the purple membrane. The protonated state of Asp-85 probably is important for the catalysis not only of all-trans <=> 13-cis thermal isomerization during dark adaptation but also of the reisomerization of the chromophore from 13-cis to all-trans configuration during N-->O-->bR transition in the photocycle. This would explain why Asp-85 stays protonated in the N and O intermediates.     

Influence of the size and protonation state of acidic residue 85 on the absorption spectrum and photoreaction of the bacteriorhodopsin chromophore.

The consequences of replacing Asp-85 with glutamate in bacteriorhodopsin, as expressed in Halobacterium sp. GRB, were investigated. Similarly to the in vitro mutated and in Escherichia coli expressed protein, the chromophore was found to exist as a mixture of blue (absorption maximum 615 nm) and red (532 nm) forms, depending on the pH. However, we found two widely separated pKa values (about 5.4 and 10.4 without added salt), arguing for two blue and two red forms in separate equilibria. Both blue and red forms of the protein are in the two-dimensional crystalline state. A single pKa, such as in the E. coli expressed protein, was observed only after solubilization with detergent. The photocycle of the blue forms was determined at pH 4.0 with 610 nm photoexcitation, and that of the red forms at pH 10.5 and with 520 nm photoexcitation, in the time-range of 100 ns to 1 s. The blue forms produced no M, but a K- and an L-like intermediate, whose spectra and kinetics resembled those of blue wild-type bacteriorhodopsin below pH 3. The red forms produced a K-like intermediate, as well as M and N. Only the red forms transported protons. Specific perturbation of the neighborhood of the Schiff base by the replacement of Asp-85 with glutamate was suggested by (1) the shift and splitting of the pKa for what is presumably the protonation of residue 85, (2) a 36 nm blue-shift in the absorption of the all-trans red chromophore and a 25 nm red-shift of the 13-cis N chromophore, as compared to wild-type bacteriorhodopsin and its N intermediate, and (3) significant acceleration of the deprotonation of the Schiff base at pH 7, but not of its reprotonation and the following steps in the photocycle.     

Chloride ion binding to bacteriorhodopsin at low pH: an infrared spectroscopic study

Bacteriorhodopsin (bR) and halorhodopsin (hR) are light-induced ion pumps in the cell membrane of Halobacterium salinarium. Under normal conditions bR is an outward proton transporter, whereas hR is an inward Cl- transporter. There is strong evidence that at very low pH and in the presence of Cl-, bR transports Cl- ions into the cell, similarly to hR. The chloride pumping activity of bR is connected to the so-called acid purple state. To account for the observed effects in bR a tentative complex counterion was suggested for the protonated Schiff base of the retinal chromophore. It would consist of three charged residues: Asp-85, Asp-212, and Arg-82. This quadruplet (including the Schiff base) would also serve as a Cl- binding site at low pH. We used Fourier transform infrared difference spectroscopy to study the structural changes during the transitions between the normal, acid blue, and acid purple states. Asp-85 and Asp-212 were shown to participate in the transitions. During the normal-to-acid blue transition, Asp-85 protonates. When the pH is further lowered in the presence of Cl-, Cl- binds and Asp-212 also protonates. The binding of Cl- and the protonation of Asp-212 occur simultaneously, but take place only when Asp-85 is already protonated. It is suggested that HCl is taken up in undissociated form in exchange for a neutral water molecule.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that Thr-46 and Thr-89 form part of a transient network of hydrogen bonds.

The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.     

Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96]

It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.     

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.     

Fourier transform infrared spectra of a late intermediate of the bacteriorhodopsin photocycle suggest transient protonation of Asp-212.

We measured time-resolved difference spectra, in the visible and the infrared, for the Glu-194 and Glu-204 mutants of bacteriorhodopsin and detected an anomalous O state, labeled O', in addition to the authentic O intermediate, before recovery of the initial state in the photocycle. The O' intermediate exhibits prominent bands at 1712 cm(-1) (positive) and 1387 cm(-1) (negative). These bands arise with the same time constant as the deprotonation of Asp-85. Both bands are shifted to lower frequency upon labeling of the protein with [4-(13)C]aspartic acid. The former band, but not the latter, is shifted in D2O. These shifts identify the two bands as the carboxyl stretch of a protonated aspartic acid and the symmetric carbonyl stretch of an unprotonated aspartate, respectively, and suggest that in O' an initially anionic aspartate enters into protonation equilibrium with Asp-85. Elimination of the few other candidates, on various grounds, identifies Asp-212 as the unknown residue. It is possible, therefore, that in the last step of the photocycle of the mutants studied the proton released from Asp-85 is conducted to the extracellular surface via Asp-212. An earlier report of a weak band at 1712 cm(-1) late in the wild-type photocycle [Zscherp and Heberle (1997) J. Phys. Chem. B 101, 10542-10547] suggests that Asp-212 might play this role in the wild-type protein also.     

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 investigation of the active site in bacteriorhodopsin: geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.     

FTIR spectroscopy of the M photointermediate in pharaonis rhoborhodopsin

pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, psR-II) is a photoreceptor for negative phototaxis in Natronobacterium pharaonis. During the photocycle of ppR, the Schiff base of the retinal chromophore is deprotonated upon formation of the M intermediate (ppR(M)). The present FTIR spectroscopy of ppR(M) revealed that the Schiff base proton is transferred to Asp-75, which corresponds to Asp-85 in a light-driven proton-pump bacteriorhodopsin (BR). In addition, the C==O stretching vibrations of Asn-105 were assigned for ppR and ppR(M). The common hydrogen-bonding alterations in Asn-105 of ppR and Asp-115 of BR were found in the process from photoisomerization (K intermediate) to the primary proton transfer (M intermediate). These results implicate similar protein structural changes between ppR and BR. However, BR(M) decays to BR(N) accompanying a proton transfer from Asp-96 to the Schiff base and largely changed protein structure. In the D96N mutant protein of BR that lacks a proton donor to the Schiff base, the N-like protein structure was observed with the deprotonated Schiff base (called M(N)) at alkaline pH. In ppR, such an N-like (M(N)-like) structure was not observed at alkaline pH, suggesting that the protein structure of the M state activates its transducer protein.     

Connectivity of the retinal Schiff base to Asp85 and Asp96 during the bacteriorhodopsin photocycle: the local-access model.

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp85 (in the extracellular direction) and Asp96 (in the cytoplasmic direction)is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp85 a proton acceptor and, subsequently, Asp96 a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L<-->M1<-->M2<-->N, and the M2-->M1 reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M1 and M2 states, but also the L and the N intermediates. Because the L and M1 states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The change in the amplitude of the C=O stretch band at 1759 cm-1 demonstrated protonation of Asp85 in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp85. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M1<-->M2 equilibration) between the two directions during the entire L-to-N segment of the photocycle.     

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.     

Effects of amino acid substitutions in the F helix of bacteriorhodopsin. Low temperature ultraviolet/visible difference spectroscopy

Site-specific mutagenesis in combination with low temperature UV/visible difference spectroscopy has been used to investigate the role of individual amino acids in the structure and function of bacteriorhodopsin (bR). We examined the effects of eight single amino acid substitutions, all in the putative F helix, on the absorption of bR as well as formation of the K and M intermediates. Both the absorbance spectra and the photocycle difference spectra of Escherichia coli expressed bR as well as the mutants S183A, P186G, and E194Q all closely resembled the corresponding purple membrane spectra. In contrast the Pro-186----Leu substitution resulted in the loss of the normal photocycle and a large blue shift in the bR state lambda max. Thus, Pro-186 appears to play a critical role in maintaining the normal protein-chromophore interactions, although the pyrrolidine ring is not essential since proline could be replaced by glycine at this position. The mutants W182F, W189F, and S193A did not appear to be directly involved in the bathochromic shift of bR since they all had lambda max's close to that of purple membrane and produced intermediates similar to K and M. However, alterations in the UV and visible difference spectra as well as the appearance of some irreversibility in the photoreactions indicate that these mutants have altered protein-chromophore interactions during the photocycle. Unlike the other mutants examined, Y185F exhibited a red-shifted form of bR and K raising the possibility that Tyr-185 is directly involved in color regulation. In addition, UV difference peaks previously associated with a tyrosine deprotonation were absent in Y185F indicating that Tyr-185 undergoes protonation changes during the photocycle in agreement with recent Fourier transform infrared difference measurements (Braiman, M.S., Mogi, T., Stern, L. J., Hackett, N., Chao, B. H., Khorana, H.G., and Rothschild, K. J. (1988) Proteins: Structure, Function, and Genetics 3, 219-229). Our results suggest that Trp-182, Tyr-185, Pro-186, Trp-189, and Ser-193, all of which are within a 100 degrees segment of the F helix, are part of a retinal-binding pocket.     

Late events in the photocycle of bacteriorhodopsin mutant L93A

In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.     

Redshift of the purple membrane absorption band and the deprotonation of tyrosine residues at high pH: Origin of the parallel photocycles of trans-bacteriorhodopsin

At high pH (> 8) the 570 nm absorption band of all-trans bacteriorhodopsin (bR) in purple membrane undergoes a small (1.5 nm) shift to longer wavelengths, which causes a maximal increase in absorption at 615 nm. The pK of the shift is 9.0 in the presence of 167 mM KCl, and its intrinsic pK is ~8.3. The red shift of the trans-bR absorption spectrum correlates with the appearance of the fast component in the light-induced L to M transition, and absorption increases at 238 and 297 nm which are apparently caused by the deprotonation of a tyrosine residue and red shift of the absorption of tryptophan residues. This suggests that the deprotonation of a tyrosine residue with an exceptionally low pK (pK_a ≈ 8.3) is responsible for the absorption shift of the chromophore band and fast M formation. The pH and salt dependent equilibrium between the two forms of bR, “neutral” and “alkaline,” bR ↔ bR_a, results in two parallel photocycles of trans-bR at high pH, differing in the rate of the L to M transition. In the pH range 10-11.8 deprotonation of two more tyrosine residues is observed with pK's ~ 10.3 and 11.3 (in 167 mM KCL). Two simple models discussing the role of the pH induced tyrosine deprotonation in the photocycle and proton pumping are presented.

It is suggested that the shifts of the absorption bands at high pH are due to the appearance of a negatively charged group inside the protein (tyrosinate) which causes electrochromic shifts of the chromophore and protein absorption bands due to the interaction with the dipole moments in the ground and excited states of bR (Stark effect). This effect gives evidence for a significant change in the dipole moment of the chromophore of bR upon excitation.

Under illumination alkaline bR forms, besides the usual photocycle intermediates, a long-lived species with absorption maximum at 500 nm (P500). P500 slowly converts into bR_a in the dark. Upon illumination P500 is transformed into an intermediate having an absorption maximum at 380 nm (P380). P380 can be reconverted to P500 by blue light illumination or by incubation in the dark.

     

Protein conformational changes in the bacteriorhodopsin photocycle

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation.Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.