Proton affinity changes driving unidirectional proton transport in the bacteriorhodopsin photocycle

Bacteriorhodopsin is the smallest autonomous light-driven proton pump. Proposals as to how it achieves the directionality of its trans-membrane proton transport fall into two categories: accessibility-switch models in which proton transfer pathways in different parts of the molecule are opened and closed during the photocycle, and affinity-switch models, which focus on changes in proton affinity of groups along the transport chain during the photocycle. Using newly available structural data, and adapting current methods of protein protonation-state prediction to the non-equilibrium case, we have calculated the relative free energies of protonation microstates of groups on the transport chain during key conformational states of the photocycle. Proton flow is modeled using accessibility limitations that do not change during the photocycle. The results show that changes in affinity (microstate energy) calculable from the structural models are sufficient to drive unidirectional proton transport without invoking an accessibility switch. Modeling studies for the N state relative to late M suggest that small structural re-arrangements in the cytoplasmic side may be enough to produce the crucial affinity change of Asp96 during N that allows it to participate in the reprotonation of the Schiff base from the cytoplasmic side. Methodologically, the work represents a conceptual advance compared to the usual calculations of pK(a) using macroscopic electrostatic models. We operate with collective states of protonation involving all key groups, rather than the individual-group pK(a) values traditionally used. When combined with state-to-state transition rules based on accessibility considerations, a model for non-equilibrium proton flow is obtained. Such methods should also be applicable to other active proton-transport systems.     

A study on the mechanism of the proton transport in bacteriorhodopsin: the importance of the water molecule

The mechanism of proton transport around the Schiff base in bacteriorhodopsin was investigated by ab initio molecular orbital (MO) calculations. Computations were performed for the case where there is a water molecule between the Schiff base and the Asp residue and for the case where there is no water molecule. Changes in the atomic configuration and potential energy through the proton transport process were compared between two cases. In the absence of water, the protonated Schiff base was not stable, and a proton was spontaneously detached from the Schiff base. On the other hand, a stable structure of the protonated Schiff base was obtained in the presence of water. This suggests that the presence of a water molecule is required for stability in the formation of a protonated Schiff base.     

Proton transfer via the external proton channel of bacteriorhodopsin

The process of proton transfer across the membrane via the external proton channel in bacteriorhodopsin is considered. A possible amino acid composition of the channel is suggested and the step-by-step mechanism of proton transfer is proposed which agrees with the experimental data. The rate of proton transfer between fixed centers at several chains of the channel was estimated for which the spectroscopic data are available.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

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.     

细菌视紫红质中质子泵出的分子机理

细菌视紫红质(Bacteriorhodopsin,或bR)是盐生嗜盐菌(Halobacterium salinarium)等细菌的跨膜蛋白质,其色基视黄醛的光致异构化作用触发细菌视紫红质的一系列结构变化,把质子从细胞质泵到细胞外空间。对细菌视紫红质中质子泵出分子机理进行了描述。     

Role of internal water molecules in bacteriorhodopsin

Internal water molecules are considered to play a crucial role in the functional processes of proton pump proteins. They may participate in hydrogen-bonding networks inside proteins that constitute proton pathways. In addition, they could participate in the switch reaction by mediating an essential proton transfer at the active site. Nevertheless, little has been known about the structure and function of internal water molecules in such proteins. Recent progress in infrared spectroscopy and X-ray crystallography provided new information on water molecules inside bacteriorhodopsin, the light-driven proton pump. The accumulated knowledge on bacteriorhodopsin in the last decade of the 20th century will lead to a realistic picture of internal water molecules at work in the 21st century. In this review, I describe how the role of water molecules has been studied in bacteriorhodopsin, and what should be known about the role of water molecules in the future.     

The effect of pH on proton transport by bacteriorhodopsin

The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.     

Proton uptake mechanism of bacteriorhodopsin as determined by time-resolved stroboscopic-FTIR-spectroscopy

Bacteriorhodopsin's proton uptake reaction mechanism in the M to BR reaction pathway was investigated by time-resolved FTIR spectroscopy under physiological conditions (293 K, pH 6.5, 1 M KCl). The time resolution of a conventional fast-scan FTIR spectrometer was improved from 10 ms to 100 μs, using the stroboscopic FTIR technique. Simultaneously, absorbance changes at 11 wavelengths in the visible between 410 and 680 nm were recorded. Global fit analysis with sums of exponentials of both the infrared and visible absorbance changes yields four apparent rate constants, k₇ = 0.3 ms, k₄ = 2.3 ms, k₃ = 6.9 ms, k₆ = 30 ms, for the M to BR reaction pathway. Although the rise of the N and O intermediates is dominated by the same apparent rate constant (k₄), protein reactions can be attributed to either the N or the O intermediate by comparison of data sets taken at 273 and 293 K. Conceptionally, the Schiff base has to be oriented in its deprotonated state from the proton donor (asp 85) to the proton acceptor (asp 96) in the M₁ to M₂ transition. However, experimentally two different M intermediates are not resolved, and M₂ and N are merged. From the results the following conclusions are drawn: (a) the main structural change of the protein backbone, indicated by amide I, amide II difference bands, takes place in the M to N (conceptionally M₂) transition. This reaction is proposed to be involved in the “reset switch” of the pump, (b) In the M to N (conceptionally M₂) transition, most likely, asp-85's carbonyl frequency shifts from 1,762 to 1,753 cm^-1 and persists in O. Protonation of asp-85 explains the red-shift of the absorbance maximum in O. (c) The catalytic proton uptake binding site asp-96 is deprotonated in the M to N transition and is reprotonated in O.     

Microsecond exchange of internal water molecules in bacteriorhodopsin

The proton-conducting pathway of bacteriorhodopsin (BR) contains at least nine internal water molecules that are thought to be key players in the proton translocation mechanism. Here, we report the results of a multinuclear (1H, 2H, 17O) magnetic relaxation dispersion (MRD) study with the primary goal of determining the rate of exchange of these internal water molecules with bulk water. This rate is of interest in current attempts to elucidate the molecular details of the proton translocation mechanism. The relevance of water exchange kinetics is underscored by recent crystallographic findings of substantial variations in the number and locations of internal water molecules during the photocycle. Moreover, internal water exchange is believed to be governed by conformational fluctuations in the protein and can therefore provide information about the thermal accessibility of functionally important conformational substates. The present 2H and 17O MRD data show that at least seven water molecules, or more if they are orientationally disordered, in BR have residence times (inverse exchange rate constant) in the range 0.1-10 micros at 277 K. At least five of these water molecules have residence times in the more restrictive range 0.1-0.5 micros. These results show that most or all of the deeply buried water molecules in BR exchange on a time-scale that is short compared to the rate-limiting step in the photocycle. The MRD measurements were performed on BR solubilized in micelles of octyl glucoside. From the MRD data, the rotational correlation time of detergent-solubilized BR was determined to 35 ns at 300 K, consistent with a monomeric protein in complex with about 150 detergent molecules. The solubilized protein was found to be stable in the dark for at least eight months at 277 K.     

Determination of the number of water molecules in the proton pathway of bacteriorhodopsin using neutron diffraction data

It has been shown that water molecules participate in the proton pathway of bacteriorhodopsin. Large efforts have been made to determine with various biophysical methods the number of water molecules involved. Neutron diffraction H2O/D2O exchange experiments have been often used to reveal the position of water even with low-resolution diffraction data. With this technique, care must be taken with the limitations of the difference Fourier method which are commonly applied to analyze the data. In this paper we compare the results of the difference Fourier method applied to measured diffraction data (not presented here) and models with those from alternative methods introduced here: (1) a computer model calculation procedure to determine a label's scattering length density based on a comparison of intensity differences derived from models and intensity differences from our measurements; (2) a method based on the Parseval formula. Both alternative methods have been evaluated and tested using results of neutron diffraction experiments on purple membranes (Hauss et al. 1994). Our findings indicate that the difference Fourier method applied to low-resolution diffraction data can successfully determine the position of localized water molecules but underestimates their integrated scattering length density in the presence of labels in other positions. Furthermore, we present the results of neutron diffraction experiments on purple membranes performed to determine the number of water molecules in the projected area of the Schiff base at 86%, 75% and 57% relative humidity (r.h.). We found 19 +/- 2 exchangeable protons at 75% r.h., which means at least 8-9 water molecules are indispensable for normal pump function.     

Kinetics of the light-driven proton movement in model membranes containing bacteriorhodopsin.

The short-circuit photoresponse of model membranes containing bacteriorhodopsin to short (35 ms) and long (3.5 s) light pulses is described. It is shown that if the light pulse is short compared with the charging and discharging times of the model membrane, the temporal response of the light-driven proton pump can be measured. Photoactive planar model membranes were formed both from biomolecular lipid membranes and from solid 6-micrometers thick Teflon septa coated with lipid and bacteriorhodopsin. The kinetic response of the pump is independent of the planar model membrane system in which it is incorporated. Experimental evidence indicates that the shape of the leading and trailing edges of the photoresponse curve for the pump deviates from simple exponential behavior. The short-circuit photoresponse of spinach chloroplast in a planar model membrane was also studied for comparison purposes.     

Light-driven proton transport of bacteriorhodopsin incorporated into long-term stable liposomes of a polymerizable sulfolipid

The chromoprotein bacteriorhodopsin from Halobacterium halobium has been incorporated into liposomes made of a fully synthetic, polymerizable lipid. Bacteriorhodopsin is found to be active in these polymer liposomes. The advantage in the use of such polymer systems concerning long-term stability in comparison with liposomes made of natural lipid is demonstrated.     

Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism.

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered lambda(max) of 555 nm and pK(a) of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.     

Lateral proton conduction along a lipid-water interface layer: a molecular mechanism for the role of hydration water molecules

Using a fluorescence method applied to phospholipids spread at the air-water interface, we showed the occurrence of a localized proton pathway along the phospholipid polar heads. From the systematic investigation of this phenomenon, we conclude that the protons diffuse through a hydrogen bond network which is a matrix of hydrated polar head groups.     

Relocation of water molecules between the Schiff base and the Thr46-Asp96 region during light-driven unidirectional proton transport by bacteriorhodopsin: an FTIR study of the N intermediate

A key event in light-driven proton pumping by bacteriorhodopsin is the formation of the L intermediate, whose transition to M is accompanied by the first proton transfer step, from the Schiff base to Asp85 on the extracellular side. Subsequent reprotonation of the Schiff base from the other side of the membrane to form the N intermediate is crucial for unidirectional proton transport. Previous FTIR studies have suggested that the intense water O-D stretching vibration bands which appear in L at 2589, 2605, and 2621 cm(-)(1) are due to a cluster of polarized water molecules connecting the Schiff base to the Thr46-Asp96 region closer to the cytoplasmic surface. In the present study the difference spectrum was obtained of the N intermediate with its photoproduct N', formed after irradiating N at 80 K. The water O-D stretching vibrations of N appear as a broad feature in a similar frequency region with a similar intensity to those of L. This feature is also affected by T46V like in L. However, the intensities of these water vibrations of N nearly returned to the initial unphotolyzed state upon formation of N', unlike those of L which are preserved in L'. An exception was V49A, which preserved the intense water vibrations of N in N'. The results suggest that both L and N have a water cluster extending from the Schiff base to Thr46. The surrounding protein moiety stabilizes the water cluster in L, but in N it is stabilized mostly by interaction with the Schiff base.