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.     

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.     

Mechanism of light-dependent proton translocation by bacteriorhodopsin.

Site-specific mutagenesis has identified amino acids involved in bR proton transport. Biophysical studies of the mutants have elucidated the roles of two membrane-embedded residues: Asp-85 serves as the acceptor for the proton from the isomerized retinylidene Schiff base, and Asp-96 participates in reprotonation of this group. The functions of Arg-82, Leu-93, Asp-212, Tyr-185, and other residues that affect bR properties when substituted are not as well understood. Structural characterization of the mutant proteins will clarify the effects of substitutions at these positions. Current efforts in the field remain directed at understanding how retinal isomerization is coupled to proton transport. In particular, there has been more emphasis on determining the structures of bR and its photointermediates. Since well-ordered crystals of bR have not been obtained, continued electron diffraction studies of purple membrane offer the best opportunity for structure refinement. Other informative techniques include solid-state nuclear magnetic resonance of isotopically labeled bR (56) and electron paramagnetic resonance of bR tagged with nitroxide spin labels (2, 3, 13, 15). Site-directed mutagenesis will be essential in these studies to introduce specific sites for derivatization with structural probes and to slow the decay of intermediates. Thus, combining molecular biology and biophysics will continue to provide solutions to fundamental problems in bR.     

Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212

Fourier transform infrared (FTIR) difference spectra have been obtained for the bR----K, bR----L, and bR----M photoreactions in bacteriorhodopsin mutants in which Asp residues 85, 96, 115, and 212 have been replaced by Asn and by Glu. Difference peaks that had previously been attributed to Asp COOH groups on the basis of isotopic labeling were absent or shifted in these mutants. In general, each COOH peak was affected strongly by mutation at only one of the four residues. Thus, it was possible to assign each peak tentatively to a particular Asp. From these assignments, a model for the proton-pumping mechanism of bR is derived, which features proton transfers among Asp-85, -96, and -212, the chromophore Schiff base, and other ionizable groups within the protein. The model can explain the observed COOH peaks in the FTIR difference spectra of bR photointermediates and could also account for other recent results on site-directed mutants of bR.     

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.     

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.     

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.     

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.     

Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle

The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied. The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium. The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin. The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate. Similar results were also obtained with the Pro-186----Gly substitution. In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate. These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.     

Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.     

Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.     

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.     

Vibrational spectroscopy of bacteriorhodopsin mutants: chromophore isomerization perturbs tryptophan-86

Fourier transform infrared difference spectra have been obtained for the bR----K and bR----M photoreactions of bacteriorhodopsin mutants with Phe replacements for Trp residues 10, 12, 80, 86, 138, 182, and 189 and Cys replacements for Trp residues 137 and 138. None of the tryptophan mutations caused a significant shift in the retinylidene C = C or C-C stretching frequencies of the visible absorption maximum of the chromophore, it is concluded that none of the tryptophan residues are essential for forming a normal bR570 chromophore. However, a 742-cm-1 negative peak attributed previously to the perturbation of a tryptophan residue during the bR----K photoreaction was found to be absent in the bR----K and bR----M difference spectra of the Trp-86 mutant. On this basis, we conclude that the structure or environment of Trp-86 is altered during the bR----K photoreaction. All of the other Trp----Phe mutants exhibited this band, although its frequency was altered in the Trp-189----Phe mutant. In addition, the Trp-182----Phe mutant exhibited much reduced formation of normal photoproducts relative to the other mutants, as well as peaks indicative of the presence of additional chromophore conformations. A model of bR is discussed in which Trp-86, Trp-182, and Trp-189 form part of a retinal binding pocket. One likely function of these tryptophan groups is to provide the structural constraints needed to prevent chromophore photoisomerization other than at the C13 = C14 double bond.     

Vibrational spectroscopy of bacteriorhodopsin mutants: evidence for the interaction of proline-186 with the retinylidene chromophore

Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue by glycine preserves the normal protein structural changes during the photocycle.     

Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F

To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.     

Bacteriorhodopsin analog regenerated with 13-desmethyl-13-iodoretinal

The retinal analog 13-desmethyl-13-iodoretinal (13-iodoretinal) was newly synthesized and incorporated into apomembranes to reconstitute bacteriorhodopsin analog 13-I-bR. The absorption maximum was 598 nm and 97% of the chromophore was an all-trans isomer in the dark- and light-adapted state. Upon flash illumination, 13-I-bR underwent a transient spectral change in which a shorter wavelength intermediate (lambda(max) = 426 nm) similar to the M species of the native bR developed. Also, 13-I-bR showed light-induced proton pumping with rates and extents comparable to those seen in the native bR. The ultraviolet circular dichroism (CD) spectrum originating from the aromatic groups was different from that of the native bR, indicating that the substituted bulky iodine atom strongly interacts with neighboring amino acids. A projection difference Fourier map showed the labeled iodine was in the vicinity of helix C. 13-I-bR is an advantageous specimen for kinetic investigations of light-induced structural changes associated with the proton pumping cycle by x-ray diffraction.     

Retinal isomerization in bacteriorhodopsin is controlled by specific chromophore-protein interactions. A study with noncovalent artificial pigments.

It has previously been shown that, in mutants lacking the Lys-216 residue, protonated Schiff bases of retinal occupy noncovalently the bacteriorhodopsin (bR) binding site. Moreover, the retinal-Lys-216 covalent bond is not a prerequisite for initiating the photochemical and proton pump activity of the pigment. In the present work, various Schiff bases of aromatic polyene chromophores were incubated with bacterioopsin to give noncovalent pigments that retain the Lys-216 residue in the binding site. It was observed that the pigment's absorption was considerably red-shifted relative to the corresponding protonated Schiff bases (PSB) in solution and was sensitive to Schiff base linkage substitution. Their PSB pK(a) is considerably elevated, similarly to those of related covalently bound pigments. However, the characteristic low-pH purple to blue transition is not observed, but rather a chromophore release from the binding site takes place that is characterized by a pK(a) of approximately 6 (sensitive to the specific complex). It is suggested that, in variance with native bR, in these complexes Asp-85 is protonated and Asp-212 serves as the sole negatively charged counterion. In contrast to the bound analogues, no photocycle could be detected. It is suggested that a specific retinal-protein geometrical arrangement in the binding site is a prerequisite for achieving the selective retinal photoisomerization.     

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.     

Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tyrosine protonation changes in bacteriorhodopsin. A Fourier transform infrared study of BR548 and its primary photoproduct

The structural alterations which occur in bacteriorhodopsin (bR) during dark adaptation (BR570----BR548) and the primary phototransition of the dark photocycle (BR548----KD610) have been investigated by Fourier transform infrared and UV difference spectroscopy. Possible contributions of tyrosine to the Fourier transform infrared difference spectra of these transitions were assigned by incorporating ring per-deuterated tyrosine into bR. Based on these data and UV difference measurements, we conclude that a stable tyrosinate exists in BR570 at physiological temperature and that it protonates during formation of BR548. A tyrosinate protonation has also been observed at low temperature during the primary phototransition of BR570 to the red-shifted photoproduct K630 (1). However, we now find that no tyrosine protonation change occurs during the primary phototransition of BR548 to the red-shifted intermediate KD610. Through analysis of bR containing isotopically labeled retinals, it was also determined that the chromophore of KD610 exits in a 13-trans, 15-cis configuration. On the basis of this evidence and previous studies on the structure of the chromophore in BR570, BR548, and K630, it appears that only the 13-trans,15-trans configuration of the protonated chromophore leads to a stable tyrosinate group. It is proposed that a tyrosinate residue is stabilized due to its interaction with the Schiff base positive charge in the BR570 chromophore. Isomerization of the chromophore about either the C13 = C14 or C = N bond disrupts this interaction causing a protonation of the tyrosinate.