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.     

Molecular dynamics study of the 13-cis form (bR548) of bacteriorhodopsin and its photocycle.

The structure and the photocycle of bacteriorhodopsin (bR) containing 13-cis,15-syn retinal, so-called bR548, has been studied by means of molecular dynamics simulations performed on the complete protein. The simulated structure of bR548 was obtained through isomerization of in situ retinal around both its C13-C14 and its C15-N bond starting from the simulated structure of bR568 described previously, containing all-trans,15-anti retinal. After a 50-ps equilibration, the resulting structure of bR548 was examined by replacing retinal by analogues with modified beta-ionone rings and comparing with respective observations. The photocycle of bR548 was simulated by inducing a rapid 13-cis,15-anti-->all-trans,15-syn isomerization through a 1-ps application of a potential that destabilizes the 13-cis isomer. The simulation resulted in structures consistent with the J, K, and L intermediates observed in the photocycle of bR548. The results offer an explanation of why an unprotonated retinal Schiff base intermediate, i.e., an M state, is not formed in the bR548 photocycle. The Schiff base nitrogen after photoisomerization of bR548 points to the intracellular rather than to the extracellular site. The simulations suggest also that leakage from the bR548 to the bR568 cycle arises due to an initial 13-cis,15-anti-->all-trans,15-anti photoisomerization.     

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are 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.     

Rotational resonance NMR study of the active site structure in bacteriorhodopsin: conformation of the Schiff base linkage.

Rotational resonance, a new solid-state NMR technique for determining internuclear distances, is used to measure a distance in the active site of bacteriorhodopsin (bR) that changes in different states of the protein. The experiments are targeted to the active site of bR through 13C labeling of both the retinal chromophore and the Lys side chains of the protein. The time course of the rotor-driven magnetization exchange between a pair of 13C nuclei is then observed to determine the dipolar coupling and therefore the internuclear distance. Using this approach, we have measured the distance from [14-13C]retinal to [epsilon-13C]Lys216 in dark-adapted bR in order to examine the structure of the retinal-protein linkage and its role in coupling the isomerizations of retinal to unidirectional proton transfer. This distance depends on the configuration of the intervening C=N bond. The 3.0 +/- 0.2 A distance observed in bR555 demonstrates that the C=N bond is syn, and the 4.1 +/- 0.3 A distance observed in bR568 demonstrates that the C=N bond is anti. These direct distance determinations independently confirm the configurations previously deduced from solid-state NMR chemical shift and resonance Raman vibrational spectra. The spectral selectivity of rotational resonance allows these two distances to be measured independently in a sample containing both bR555 and bR568; the presence of both states and of 25% lipid in the sample demonstrates the use of rotational resonance to measure an active site distance in a membrane protein with an effective molecular mass of about 85 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity effects in the binding of all-trans retinal to bacterio-opsin

The special trimeric structure of bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarum, and especially, the still controversial question as to whether the three protein components are structurally and functionally identical, have been subject to considerable work. In the present work, the problem is approached by studying the reconstitution reaction of the bR apo-protein with all-trans retinal, paying special attention to the effects of the apo-protein/retinal (P:R) ratio. The basic observation is that at high P:R values, the reconstitution reaction proceeds via two distinct, fast and slow, pathways associated with two different pre-pigment precursors absorbing at 430 nm (P(430)) and 400 nm (P(400)), respectively. These two reactions, exhibiting 2:1 (P(430)/P(400)) amplitude ratios, are markedly affected by the P:R value. The principal feature is the acceleration of the P(400) --> bR transition at low P:R ratios. The data are interpreted in terms of a scheme in which the added retinal first occupies two protein retinal traps, R(1) and R(2), from which it is transferred to two spectroscopically distinct binding sites corresponding to the two pre-pigments, P(430) and P(400), respectively. Two noncovalently bound retinal molecules occupy two P(430) sites of the bR trimer, while one (P(400)) occupies the third. Binding is completed by generating the retinal-protein covalent bond. Analogous experiments were also carried out with an aromatic bR chromophore and with the D85N bR mutant. The accumulated data clearly point out the heterogeneity of the binding reaction intermediates, in which two are clearly distinct from the third. However, CD spectroscopy strongly suggests that even the two P(430) sites are not structurally identical. The heterogeneity of the P intermediates in the binding reaction can be accounted for, either by being induced by cooperativity or by an intrinsic heterogeneity that is already present in the apoprotein. The question as to whether the final reconstituted pigment, as well as native bR, are nonhomogeneous should be the subject of future studies.     

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.     

Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect.     

Ultrafast spectroscopy of biological photoreceptors

We review recent new insights on reaction dynamics of photoreceptors proteins gained from ultrafast spectroscopy. In Blue Light sensing Using FAD (BLUF) domains, a hydrogen-bond rearrangement around the flavin chromophore proceeds through a radical-pair mechanism, by which light-induced electron and proton transfer from the protein to flavin result in rotation of a conserved glutamine that switches the hydrogen bond network. Femtosecond infrared spectroscopy has shown that in photoactive yellow protein (PYP), breaking of a hydrogen bond that connects the p-coumaric acid chromophore to the backbone is crucial for trans-cis isomerization and successful entry into the photocycle. Furthermore, isomerization reactions of phycocyanobilin in phytochrome and retinal in the rhodopsins have been revealed in detail through application of femtosecond infrared and femtosecond-stimulated Raman spectroscopy.     

Tracing cytoplasmic Ca(2+) ion and water access points in the Ca(2+)-ATPase

Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) transports two Ca(2+) ions across the membrane of the sarco(endo)plasmic reticulum against the concentration gradient, harvesting the required energy by hydrolyzing one ATP molecule during each transport cycle. Although SERCA is one of the best structurally characterized membrane transporters, it is still largely unknown how the transported Ca(2+) ions reach their transmembrane binding sites in SERCA from the cytoplasmic side. Here, we performed extended all-atom molecular dynamics simulations of SERCA. The calculated electrostatic potential of the protein reveals a putative mechanism by which cations may be attracted to and bind to the Ca(2+)-free state of the transporter. Additional molecular dynamics simulations performed on a Ca(2+)-bound state of SERCA reveal a water-filled pathway that may be used by the Ca(2+) ions to reach their buried binding sites from the cytoplasm. Finally, several residues that are involved in attracting and guiding the cations toward the possible entry channel are identified. The results point to a single Ca(2+) entry site close to the kinked part of the first transmembrane helix, in a region loaded with negatively charged residues. From this point, a water pathway outlines a putative Ca(2+) translocation pathway toward the transmembrane ion-binding sites.     

Molecular dynamics study of early picosecond events in the bacteriorhodopsin photocycle: dielectric response, vibrational cooling and the J, K intermediates.

Molecular dynamics simulations have been carried out to study the J625 and K590 intermediates of bacteriorhodopsin's (bRs) photocycle starting from a refined structure of bR568. The coupling between the electronic states of retinal and the protein matrix is characterized by the energy difference delta E(t) between the excited state and the ground state to which the protein contributes through the Coulomb interaction. Our simulations indicate that the J625 intermediate is related to a polarization of the protein matrix due to the brief (200 fs) change of retinal's charge distribution in going to the excited state and back to the ground state, and that the rise time of the K590 intermediate is determined by vibrational cooling of retinal.     

Escape of a Small Molecule from Inside T4 Lysozyme by Multiple Pathways

The T4 lysozyme L99A mutant is often used as a model system to study small-molecule binding to proteins, but pathways for ligand entry and exit from the buried binding site and the associated protein conformational changes have not been fully resolved. Here, molecular dynamics simulations were employed to model benzene exit from its binding cavity using the weighted ensemble (WE) approach to enhance sampling of low-probability unbinding trajectories. Independent WE simulations revealed four pathways for benzene exit, which correspond to transient tunnels spontaneously formed in previous simulations of apo T4 lysozyme. Thus, benzene unbinding occurs through multiple pathways partially created by intrinsic protein structural fluctuations. Motions of several α-helices and side chains were involved in ligand escape from metastable microstates. WE simulations also provided preliminary estimates of rate constants for each exit pathway. These results complement previous works and provide a semiquantitative characterization of pathway heterogeneity for binding of small molecules to proteins.     

New insights into the effect of mutations on affibody-Fc interaction,a molecular dynamics simulation approach

Staphylococcal protein A (SpA) domain B (the basis of affibody) has been widely used in affinity chromatography and found therapeutic applications against inflammatory diseases through targeting the Fc part of immunoglobulin G (IgG). We have performed extensive molecular dynamics simulation of 41 SpA mutants and compared their dynamics and conformations to wild type. The simulations revealed the molecular details of structural and dynamics changes that occurred due to introducing point mutations and helped to explain the SPR results. It was observed in some variants a point mutation caused extensive structural changes far from the mutation site, while an effect of some other mutations was limited to the site of the mutated residue. Also, the pattern of hydrogen bond networks and hydrophobic core arrangements were investigated. We figured out mutations that occurred at positions 128, 136, 150 and 153, affected two hydrophobic cores at the interface as well as mutations introduced at positions 129 and 154 interrupted two hydrogen bond networks of the interface, SPR data showed all of these mutations reduced binding affinity significantly. Overall, by scanning the SpA-Fc interface through the large numbers of introduced mutations, the new insights have been gained which would help to design high- affinity ligands of IgG.     

Steered molecular dynamics simulations on the "tail helix latch" hypothesis in the gelsolin activation process

The molecular basis of the "tail helix latch" hypothesis in the gelsolin activation process has been studied by using the steered molecular dynamics simulations. In the present nanosecond scale simulations, the tail helix of gelsolin was pulled away from the S2 binding surface, and the required forces were calculated, from which the properties of binding between the tail helix and S2 domain and their dynamic unbinding processes were obtained. The force profile provides a detailed rupture mechanism that includes six major unbinding steps. In particular, the hydrogen bonds formed between Arg-207 and Asp-744 and between Arg-221 and Leu-753 are of the most important interaction pairs. The two hydrogen bond "clamps" stabilize the complex. The subsequent simulation on Arg-207-Ala (R207A) mutation of gelsolin indicated that this mutation facilitates the unbinding of the tail helix and that the contribution of the hydrogen bond between Arg-207 and Asp-744 to the binding is more than 50%, which offers a new clue for further mutagenesis study on the activation mechanism of gelsolin. Surrounding water molecules enhance the stability of the tail helix and facilitate the rupture process. Additionally, temperature also has a significant effect on the conformation of the arginine and arginine-related interactions, which revealed the molecular basis of the temperature dependence in gelsolin activation.     

Consequences of breaking the Asp-His hydrogen bond of the catalytic triad: effects on the structure and dynamics of the serine esterase cutinase.

The objective of this study has been to investigate the effects on the structure and dynamics that take place with the breaking of the Asp-His hydrogen bond in the catalytic triad Asp175-His188-Ser120 of the serine esterase cutinase in the ground state. Four molecular dynamics simulations were performed on this enzyme in solution. The starting structures in two simulations had the Asp175-His188 hydrogen bond intact, and in two simulations the Asp175-His188 hydrogen bond was broken. Conformations of the residues comprising the catalytic triad are well behaved during both simulations containing the intact Asp175-His188 hydrogen bond. Short contacts of less than 2.6 A were observed in 1.2% of the sampled distances between the carboxylate oxygens of Asp175 and the NE2 of His188. The simulations showed that the active site residues exhibit a great deal of mobility when the Asp175-His188 hydrogen bond is broken. In the two simulations in which the Asp175-His188 hydrogen bond is not present, the final geometries for the residues in the catalytic triad are not in catalytically productive conformations. In both simulations, Asp175 and His188 are more than 6 A apart in the final structure from dynamics, and the side chains of Ser120 and Asp175 are in closer proximity to the NE2 of His188 than to ND1. Nonlocal effects on the structure of cutinase were observed. A loop formed by residues 26-31, which is on the opposite end of the protein relative to the active site, was greatly affected. Further changes in the dynamics of cutinase were determined from quasiharmonic mode analysis. The frequency of the second lowest mode was greatly reduced when the Asp175-His188 hydrogen bond was broken, and several higher modes showed lower frequencies. All four simulations showed that the oxyanion hole, composed of residues Ser42 and Gln121, is stable. Only one of the hydrogen bonds (Ser42 OG to Gln121 NE2) observed in the crystal structure that stabilize the conformation of Ser42 OG persisted throughout the simulations. This hydrogen bond appears to be enough for the oxyanion hole to retain its structural integrity.     

Influence of the disulfide bond configuration on the dynamics of the spin label attached to cytochrome c

A series of multi-nanosecond molecular dynamics (MD) simulations of wild-type cytochrome c and its spin-labeled variants with the methanethiosulfonate moiety attached at position C102 were performed (1) to elucidate the effect of the spin probe presence on the protein structure and (2) to describe the structure and dynamics of the spin-label moiety. Comparisons with the reference crystal structure of cytochrome c (PDB entry: 1YCC) indicate that the protein secondary structure is well preserved during simulations of the wild-type cytochrome c but slightly changed in simulations of the cytochrome c labeled at position C102. At the time scale covered in our simulations, the spin label exhibits highly dynamical behavior. The number of observed distinct conformations of the spin label moiety is between 3 and 13. The spin probe was found to form short-lived hydrogen bonds with the protein. Temporary hydrophobic interactions between the probe and the protein were also detected. The MD simulations directly show that the disulfide bond in the tether linking a spin probe with a protein strongly influence the behavior of the nitroxide group. The conformational flexibility and interaction with the protein are different for each of the two low energy conformations of the disulfide bond.     

Specific binding sites for cations in bacteriorhodopsin.

The Asp-85 residue, located in the vicinity of the retinal chromophore, plays a key role in the function of bacteriorhodopsin (bR) as a light-driven proton pump. In the unphotolyzed pigment the protonation of Asp-85 is responsible for the transition from the purple form (lambda(max) = 570 nm) to the blue form (lambda(max) = 605 nm) of bR. This transition can also be induced by deionization (cation removal). It was previously proposed that the cations bind to the bR surface and raise the surface pH, or bind to a specific site in the protein, probably in the retinal vicinity. We have reexamined these possibilities by evaluating the interaction between Mn(2+) and a nitroxyl radical probe covalently bound to several mutants in which protein residues were substituted by cystein. We have found that Mn(2+), which binds to the highest-affinity binding site, significantly affects the EPR spectrum of a spin label attached to residue 74C. Therefore, it is concluded that the highest-affinity binding site is located in the extracellular side of the protein and its distance from the spin label at 74C is estimated to be approximately 9.8 +/- 0.7 A. At least part of the three to four low-affinity cation binding sites are located in the cytoplasmic side, because Mn(2+) bound to these binding sites affects spin labels attached to residues 103C and 163C located in the cytoplasmic side of the protein. The results indicate specific binding sites for the color-controlling cations, and suggest that the binding sites involve negatively charged lipids located on the exterior of the bR trimer structure.     

Redox potentials of the oriented film of the wild-type,the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were -470 mV for the 13-cis configuration of the retinal Shiff base in bR and -757 mV for the all-trans configuration in H(2)O, and -433 mV for the 13-cis configuration and -742 mV for the all-trans configuration in D(2)O. The solvent isotope effect (DeltaV=V(D(2)O)-V(H(2)O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated C=N part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were -507 mV for the 13-cis configuration and -788 mV for the all-trans configuration; and for the E204Q mutant they were -491 mV for the 13-cis configuration and -769 mV for the all-trans configuration. Replacement of the Glu(194) or Glu(204) residues by Gln weakened the electron withdrawing interaction to the protonated C=N bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were -471 mV for the 13-cis configuration and -760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the C=N part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.     

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.