Regeneration of Bacteriorhodopsin from Thermally Unfolded Bacterio-Opsin and All-trans Retinal at High Temperatures

The temperature dependence of regeneration of bacteriorhodopsin (bR) from its apoprotein, bacterio-opsin (bO), and all-trans retinal was investigated using two different procedures to probe the structural properties of bO at high temperatures. Regeneration experiments performed at 25 °C after incubation of bO within the temperature range of 35–75 °C indicate that irreversible thermal unfolding begins at 50 °C. When bO is incubated for one hour and mixed with retinal at the same elevated temperatures, however, a greater extent of regeneration to bR occurs, even at temperatures ranging from 50 to 65 °C. These experimental results indicate that regeneration of bR occurs from thermally unfolded bO and suggest dynamic structural fluctuation of bO in the unfolded state.     

Heterogeneity in regeneration of bacteriorhodopsin from bacterio-opsin and all-trans retinal at high temperatures: implications for dynamic structural fluctuations

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 degrees C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.     

Heterogeneity in Regeneration of Bacteriorhodopsin from Bacterio-Opsin and All-trans Retinal at High Temperatures: Implications for Dynamic Structural Fluctuations

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 °C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.     

Irreversible conformational change of bacterio-opsin induced by binding of retinal during its reconstitution to bacteriorhodopsin, as studied by (13)C NMR

We compared (13)C NMR spectra of [3-(13)C]Ala- and [1-(13)C]Val-labeled bacterio-opsin (bO), produced either by bleaching bR with hydroxylamine or from a retinal-deficient strain, with those of bacteriorhodopsin (bR), in order to gain insight into the conformational changes of the protein backbone that lead to correct folding after retinal is added to bO. The observed (13)C NMR spectrum of bO produced by bleaching is not greatly different from that of bR, except for the presence of suppressed or decreased peak-intensities. From careful evaluation of the intensity differences between cross polarization magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) spectra, it appears that the reduced peak-intensities arise from reduced efficiency of cross polarization or interference of internal motions with proton decoupling frequencies. In particular, the E-F and F-G loops and some transmembrane helices of the bleached bO have acquired internal motions whose frequencies interfere with proton decoupling frequencies. In contrast, the protein backbone of the bO from the retinal-negative cells is incompletely folded. Although it contains mainly a-helices, its very broad (13)C NMR signals indicate that its tertiary structure is different from bR. Importantly, this changed structure is identical in form to that of bleached bO from wild-type bR after it was regenerated with retinal in vitro, and bleached with hydroxylamine. We conclude that the binding of retinal is essential for the correct folding of bR after it is inserted in vitro into the lipid bilayer, and the final folded state does not revert to the partially folded form upon removal of the retinal.     

Decay of the tryptophan fluorescence anisotropy in bacteriorhodopsin and its modified forms.

In this work we study the decay of the polarization of the Trp fluorescence in native bacteriorhodopsin (bR), deionized bR (dlbR), and the retinal-free form of bR, bacterioopsin (bO), using picosecond laser/streak camera system. Two types of depolarization processes are observed, one around 250 ps, which is temperature independent around room temperature, and the other in the 1-3-ns range, which is sensitive to temperature and certain bR modifications. This suggests the presence of at least two different environments for the eight Trp molecules in bR. Native bR and deionized bR gave the same depolarization decay times, suggesting that the removal of metal cations does not change the microenvironment of the emitting Trp molecules. The slow component is faster in bO than in bR, suggesting a change in the environment of the Trp molecules upon the removal of the retinal chromophore. All these results are discussed in terms of the different mechanisms of Trp fluorescence depolarization. A comparison between the depolarization decay in rhodopsin and bR is made.     

Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin.

Bacteriorhodopsin (bR) is a light-driven proton pump from Halobacterium salinarium and is a model system for studying membrane protein folding, stability, function, and structure. bR is composed of bacterio-opsin (bO), the 248-amino acid apo protein, and all-trans retinal, which is linked to lysine 216 via a protonated Schiff base. A bO gene (sbOd) possessing 29 unique restriction sites and a carboxyl-terminal purification epitope (1D4, nine amino acids) has been designed and synthesized. Overexpression of bO was achieved by fusion to the carboxyl terminus of maltose binding protein (MBP). The expressed fusion protein (MBP-sbO-1D4) formed inclusion bodies in Escherichia coli and, following solubilization with urea and removal of the urea by dialysis, approximately 170 mg of approximately 75% pure MBP-sbO-1D4 was obtained from 1 L of culture. MBP-sbO-1D4 formed high molecular weight (> or = 2,000 kDa) oligomers that were water-soluble. The synthetic bO with the 1D4 tag (sbO-1D4) was separated from MBP by trypsin cleavage at the factor Xa site between the MBP and sbO-1D4 domains. Selective trypsin cleavage at the factor Xa site, instead of at the 14 other potential trypsin sites within bO, was accomplished by optimization of the digestion conditions. Both MBP-sbO-1D4 and sbO-1D4 were regenerated with all-trans retinal and purified to homogeneity. In general, 6-10 mg of sbR-1D4 and 52 mg of MBP-sbR-1D4 were obtained from 1 L of cell culture. No significant differences in terms of UV/vis light absorbance, light/dark adaptation, and photocycle properties were observed among sbR-1D4, MBP-sbR-1D4, and bR from H. salinarium.     

Thermodynamic stability of bacteriorhodopsin mutants measured relative to the bacterioopsin unfolded state

The stability of bacteriorhodopsin (bR) has often been assessed using SDS unfolding assays that monitor the transition of folded bR (bR(f)) to unfolded (bR(u)). While many criteria suggest that the unfolding curves reflect thermodynamic stability, slow retinal (RET) hydrolysis during refolding makes it impossible to perform the most rigorous test for equilibrium, i.e., superimposable unfolding and refolding curves. Here we made a new equilibrium test by asking whether the refolding rate in the transition zone is faster than RET hydrolysis. We find that under conditions we have used previously, refolding is in fact slower than hydrolysis, strongly suggesting that equilibrium is not achieved. Instead, the apparent free energy values reported previously are dominated by unfolding rates. To assess how different the true equilibrium values are, we employed an alternative method by measuring the transition of bR(f) to unfolded bacterioopsin (bO(u)), the RET-free form of unfolded protein. The bR(f)-to-bO(u) transition is fully reversible, particular when we add excess RET. We compared the difference in unfolding free energies for 13 bR mutants measured by both assays. For 12 of the 13 mutants with a wide range of stabilities, the results are essentially the same within experimental error. The congruence of the results is fortuitous and suggests the energetic effects of most mutations may be focused on the folded state. The bR(f)-to-bO(u) reaction is inconvenient because many days are required to reach equilibrium, but it is the preferable measure of thermodynamic stability. This article is part of a Special Issue entitled: Protein Folding in Membranes.     

Effect of temperature, pH, and metal ion binding on the secondary structure of bacteriorhodopsin: FT-IR study of the melting and premelting transition temperatures

We have measured the temperature dependence of the FT-IR spectra of bacteriorhodopsin (bR) as a function of the pH and of the divalent cation regeneration with Ca(2+) and Mg(2+). It has been found that although the irreversible melting transition shows a strong dependence on the pH of the native bR, the premelting reversible transition at 78-80 degrees C shows very little variation over the pH range studied. It is further shown that the acid blue bR shows a red-shifted amide I spectrum at physiological temperature, which shows a more typical alpha-helical frequency component at 1652 cm(-)(1) and could be the reason for the observed reduction of its melting temperature and lack of an observed premelting transition. Furthermore, the thermal transitions for Ca(2+)- and Mg(2+)-regenerated bR (Ca-bR and Mg-bR, respectively) each show a premelting transition at the same 78-80 degrees C temperature as the native purple membrane, but the irreversible melting transition has a slight dependence on the cation identity. The pH dependence of the Ca(2+)-regenerated bR is studied, and neither transition varies over the pH range studied. These results are discussed in terms of the cation contribution to the secondary structural stability in bR.     

Effect of transmembrane helix packing on tryptophan and tyrosine environments in detergent-solubilized bacterio-opsin

Bacterio-opsin (bO) is folded in a nearly native conformation in mixed micelles of dimyristoyl phosphatidyl choline (DMPC) and 3-[(3-cholamidopropyl)-dimehtylamonio]-1-propane sulfonic acid (CHAPS), but bO is partially unfolded in sodium dodecyl sulfate (SDS). UV difference spectroscopy was used to study the changes in environment of bO aromatic amino acid side chains that occur upon partial unfolding. The UV difference spectra of peptides in CHAPS/DMPC minus peptides in SDS were measured for bO and the following subfragments of bO: C1 (residues 72–248), C2 (1–71), V1 (1–166), V2 (167–248), CB7 (119–145), CB9 (164–209), and CB10 (72–118). The spectra show that, in partially unfolded bO in SDS, the Tyr and Trp absorbance is blue-shifted. The difference spectra were compared to solvent perturbation difference spectra of N-acetyl-l-tyrosine ethyl ester and N-acetyl-l-tryptophanamide. The exposure change calculated from the difference spectra was found to correlate with the change in the number of van der Waals contacting atoms upon partial unfolding, and also with the number of transmembrane helical segments. This result suggests a simple experimental method of testing helix packing arrangements derived from hydropathy plots and model building.Abbreviations bO bacterio-opsin - bR bacteriorhodopsin - SDS sodium dodecyl sulfate - CHAPS 3-[(3-cholamidopropyl)-dimethylamonio]-1-propane sulfonic acid - C1 bacteriorhodopsin residues 72–248 - C2 1-71 - VI 1-166 - V2 167-248 - CB7 119-145 - CB9 164-209 - CB10 72-118 - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine - EDTA di-sodium ethylenediamine tetra-aceticacid     

The role of the native lipids and lattice structure in bacteriorhodopsin protein conformation and stability as studied by temperature-dependent Fourier transform-infrared spectroscopy

We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65 degrees C, and the pre-melting transition in which the protein changes conformation in native bR at 80 degrees C could not be observed. Also, the N[bond]H to N[bond]D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91 degrees C compared with 80 degrees C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91 degrees C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.     

Fourier transform infrared study of the effect of different cations on bacteriorhodopsin protein thermal stability

The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca(2+), and Mg(2+), which we studied previously, are compared with Mn(2+), Hg(2+), and a large, synthesized divalent organic cation, ((Et)(3)N)(2)Bu(2+). It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652 cm(-1). This always occurs at approximately 80 degrees C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623 cm(-1), is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR.     

Helix formation and the unfolded state of a 52-residue helical protein

A growing class of proteins in biological processes has been found to be unfolded on isolation under normal solution conditions. We have used NMR spectroscopy to characterize the structural and dynamic properties of the unfolded and partially folded states of a 52-residue alanine-rich protein (Ala-14) at temperatures from -5 degrees C to 40 degrees C. At 40 degrees C, alanine residues in Ala-14 adopt phi and psi angles, consistent with a significant ensemble population of polyproline II conformation. Analysis of relaxation rates in the protein reveals that a series of residues, Gln 35-Ala 36-Ala 37-Lys 38-Asp 39-Asp 40-Ala 41-Ala 42, displays slow motional dynamics at both -5 degrees C and 40 degrees C. Temperature-dependent chemical shift changes indicate that this region is the site of helix initiation. The remaining N-terminal residues become increasingly dynamic as they extend from the nucleation site. The C terminus remains dynamic and changes less with temperature, indicating it is relatively unstructured. Ala-14 provides a high-resolution portrait of the unfolded state and the process of helix nucleation and propagation in the absence of tertiary contacts, information that bears on early events in protein folding.     

The effect of protein conformation change from alpha(II) to alpha(I) on the bacteriorhodopsin photocycle

The bacteriorhodopsin (bR) photocycle was followed by use of time-resolved Fourier-transform infrared (FTIR) spectroscopy as a function of temperature (15-85 degrees C) as the alpha(II) --> alpha(I) conformational transition occurs. The photocycle rate increases with increasing temperature, but its efficiency is found to be drastically reduced as the transition takes place. A large shift is observed in the all-trans left arrow over right arrow 13-cis equilibrium due to the increased stability of the 13-cis isomer in alpha(I) form. This, together with the increase in the rate of dark adaptation as the temperature increases, leads to a large increase in the 13-cis isomer concentration in bR in the alpha(I) form. The fact that 13-cis retinal has a much-reduced absorption cross-section and its inability to pump protons leads to an observed large reduction in the concentration of the observed photocycle intermediates, as well as the proton gradient at a given light intensity. These results suggest that nature might have selected the alpha(II) rather than the alpha(I) form as the helical conformation in bR to stabilize the all-trans retinal isomer that is a better light absorber and is capable of pumping protons.     

Protein-DNA binding correlates with structural thermostability for the full-length human p53 protein

Full-length p53 protein purified from Escherichia coli in the unmodified, "latent" form was examined by several methods to correlate thermal stability of structure with functional DNA binding. Structure prediction algorithms indicate that the majority of beta-sheet structure occurs in the p53 core DNA binding domain. Circular dichroism spectra demonstrate that the intact protein is surprisingly stable with a midpoint for the irreversible unfolding transition at approximately 73 degrees C. Significant beta-sheet structural signal remains even to 100 degrees C. The persistent beta-sheet CD signal correlates with significant DNA binding (K(d) approximately nM range) to temperatures as high as 50 degrees C. These data confirm the ability of the DNA binding domain in the full-length "latent" protein to bind consensus dsDNA targets effectively in the absence of activators over a broad temperature range. In addition, we demonstrate that Ab1620 reactivity is not directly correlated with the functional activity of the full-length protein since loss of this epitope occurs at temperatures at which significant specific DNA binding can still be measured.     

Primary intermediates in the photochemical cycle of bacteriorhodopsin.

Picosecond studies of the primary photochemical events in the light-adapted bacteriorhodopsin, bR570, indicate that the first metastable intermediate K610 is formed with a rise time of 11 ps. Difference spectra obtained at 50 ps after excitation show that K610 is the same species as that trapped in low temperature glasses. A precursor species (S) of the K610 intermediate has been observed which is red shifted with respect to K610 and is formed within the 6-ps time width of the excitation pulse. The formation of the precursor has no observable thermal dependence between 298 degrees and 1.8 degrees K. The formation of K610 has a very low thermal barrier and at very low temperatures, the rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation becomes practically temperature independent which is characteristic of a tunneling process. The rate of formation of K610 has a moderate deuterium isotope effect of kH/kD approximately 1.6 at 298 degrees K and 2.4 at 4 degrees K. The mechanism for formation of K610 is found to involve a rate-limiting proton transfer which occurs by tunneling at low temperatures.     

Time-resolved resonance Raman characterization of the bO640 intermediate of bacteriorhodopsin. Reprotonation of the Schiff base.

The resonance Raman spectrum of photolyzed bacteriorhodopsin under conditions known to increase the concentration of the bO640 intermediate in both H2O and D2O is presented. By use of computer subtraction techniques and a knowledge of the Raman spectra of the unphotolyzed bacteriorhodopsin as well as the other intermediates in the cycle, a qualitative spectrum of bO640 is determined. The shift of a band at 1630 cm-1 in H2O to 1616 cm-1 in D2O suggests that the Schiff base of bO640 is protonated. Additional bands at 947, 965, and 992 cm-1 that appear only in D2O suspensions confirm that a proton is coupled to the retinal chromophore of bO640. The reprotonation of the Schiff base thus occurs during the bM412 to bO640 step. The fingerprint region, sensitive to the isomeric configuration of the retinal chromophore of bO640, is dissimilar to the fingerprint regions of published model compounds and other forms of bacteriorhodopsin.     

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.     

Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.

Solid-state 13C NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-13C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80 degrees C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic investigatons on the structural thermal stability of leucine aminopeptidase. ESR, CD and fluorescence studies]

The thermal behaviour of leucine aminopeptidase (LAP, EC: 3.4.11.1) from bovine eye lens has been investigated in the temperature region 20--70 degrees C by spin-labelling of SH-groups (ESR), by CD and by fluorescence of tryptophane residues. Enzymatic activity of LAP was compared with spectroscopic data in this temperature region. From 20-60 degrees C the structural parts (alpha, beta, random coil) estimated from CD spectra remain unchanged. Within 20-55 degrees C no irreversible exposure of tryptophane residues takes place. In both types of spin-labelled LAP the strong immobilizing environment of the label retains its highly ordered structure up to 55 degrees C. Reversible changes of mobility and polarity of the environment of the label induced by temperature within 20-50 degrees C do not reduce the enzymatic activity and are regarded as local loosening of ordered structure. At 65 degrees C strong precipitation occurs. From 55 degrees C to 65 degrees C tryptophane residues are irreversibly exposed. The highly ordered environment of the label is destroyed about 55 degrees C, and a considerable amount of spin label molecules is reduced at the NO group by exposed SH groups. The above mentioned local loosening of structure becomes irreversible at 60 degrees C. The environment of both labels dominating above 60 degrees C is highly mobile and strongly polar and represents an extensively unfolded conformation. Until 60 degrees C no essential disordering of protein structure leading to a decrease of enzymatic activity occurs. Above 60 degrees C a sharp breakdown of ordered structures takes place, which is accompanied by a strong diminution of enzymatic activity.     

Low temperature FTIR study of the Schiff base reprotonation during the M-to-bR backphotoreaction: Asp 85 reprotonates two distinct types of Schiff base species at different temperatures

We have applied low temperature difference FTIR spectroscopy to investigate intermediates produced from the M intermediate upon blue light excitation (<480 nm). In agreement with an earlier report by Balashov and Litvin (1981), who studied these intermediates with low temperature visible absorption spectrophotometry, we have observed at least three stages in this backphotoreaction. The initial photoproduct is stable at 100 K, and two products of subsequent thermal reactions are observed upon raising the temperature to 130 and 160 K, respectively.

The alterations in the C=N stretching mode of the Schiff base have been identified by isotopically labeling the retinal chromophore, and changes in C=O stretching modes of amino acid residues with acidic side chains have been investigated. Analysis of the C=N stretching mode shows that the Schiff base remains unprotonated after the photochemical reaction at 100 K. Moreover, there are two types of Schiff bases, presumably associated with different bR species, that become thermally reprotonated at 130 and 160 K, respectively. Bands associated with the C=O stretching modes suggest that Asp 85 rather than Asp 96 reprotonates the Schiff base during the M to bR backphotoreaction. This conclusion is consistent with earlier observations that the polarity of electrical signals during this photochemical back reaction is reversed as compared to the thermal regeneration of bR from M.