Protonation dynamics of the extracellular and cytoplasmic surface of bacteriorhodopsin in the purple membrane (original) (raw)
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MOLECULAR ASPECTS OF LIGHT-INDUCED UPTAKE AND RELEASE OF PROTONS BY PURPLE MEMBRANES
Photochemistry and Photobiology, 1981
The precise molecular description of the time dependent steps in the uptake, translocation and release of protons by bacteriorhodopsin following photon absorption requires information on the time resolved changes in protonation of the side chains of specific amino acid residues and the correlation of these changes with photocycle kinetics. Thus far, the use of chemical modification to probe the role of amino acid side chains in this process has proven of value in demonstrating a role for tyrosine residues in release and uptake of protons associated with early and later stages (before and after M412 formation) of the photocycle. In addition, it has demonstrated the essential role of ionic interactions between negatively charged carboxyl groups and positively charged guanidinium groups of arginine, and amino groups of lysine. The transmembrane regulatory effect of ApH+ on the M412 species of the photocycle provides additional evidence for the participation of reversible protonation of amino acid side chains at the surfaces of the purple membrane in the mechanism of proton translocation. Thus, our studies relate molecular events of proton translocation to the bioenergetics of the purple membrane.
Biochemistry, 1997
Bacteriorhodopsin is the light-driven proton-pumping protein of Halobacterium salinarum that extracts protons from the well-buffered cytoplasmic space within the time limits set by the photocycle turnover. The specific mechanism of the proton uptake by the cytoplasmic surface of the protein was investigated in this study by the laser-induced proton pulse technique. The purple membrane preparations were labeled by fluorescein at two residues (36 or 38) of the cytoplasmic surface of the protein, sites that are close to the orifice of the proton-conducting channel. The membranes were pulsed by protons discharged from photoexcited pyranine [
Proton Transfer Dynamics on the Surface of the Late M State of Bacteriorhodopsin
Biophysical Journal, 2002
The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sassmodel that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state.
Journal of The American Chemical Society, 2006
Proton binding and release are elementary steps for the transfer of protons within proteins, which is a process that is crucial in biochemical catalysis and biological energy transduction. Local electric fields in proteins affect the proton binding energy compared to aqueous solution. In membrane proteins, also the membrane potential affects the local electrostatics and can thus be crucial for protein function. In this paper, we introduce a procedure to calculate the protonation probability of titratable sites of a membrane protein in the presence of a membrane potential. In the framework of continuum electrostatics, we use a modified Poisson-Boltzmann equation to include the influence of the membrane potential. Our method considers that in a transmembrane protein each titratable site is accessible for protons from only one side of the membrane depending on the hydrogen bond pattern of the protein. We show that the protonation of sites receiving their protons from different sides of the membrane is differently influenced by the membrane potential. In addition, the effect of the membrane potential is combined with the effect of the pH gradient resulting from proton pumping. Our method is applied to bacteriorhodopsin, a light-activated proton pump. We find that the proton pumping of this protein might be regulated by Asp115, a conserved residue for which no function has been identified yet. According to our calculations, the interaction of Asp115 with Asp85 leads to the protonation of the latter if the pH gradient or the membrane potential becomes too large. Since Asp85 is the primary proton acceptor in the photocycle, bacteriorhodopsin molecules in which Asp85 is protonated cannot pump protons. Furthermore, we estimate how the membrane potential affects the energetics of the individual proton-transfer reactions of the photocycle. Most reactions, except the initial proton transfer from the Schiff base to Asp85, are influenced. Our calculations give new insights into the mechanism with which bacteriorhodopsin senses the membrane potential and the pH gradient and how the proton pumping is regulated by these parameters.
The quantum efficiency of proton pumping by the purple membrane of Halobacterium halobium
Biophysical Journal, 1980
The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Schiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is -1.8 in 0.5 M KCI and -0.64 in 10 mM KCI. Direct measurements of the quantum yield of H+ give -0.7 when the PM is suspended in 0.5 M KCI and 0.25 in 10 mM KCI. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977. Biophys. J. 17:185.), these measurements also give a H+/M412 -2 at high salt. In PM/PC vesicles, the H+/M412 is -2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Additon of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.
Proton transfer dynamics at membrane/water interface and mechanism of biological energy conversion
Biochemistry (Moscow), 2005
A retrospective account of studies on proton transfer dynamics at the membrane surface might be an appropri ate contribution to this special issue in honor of Vladimir Skulachev, who has published seminal works in this field. Below we focus on the mechanisms of proton transfer both across and along the membrane/water interface as inferred from pulse experiments with light triggered enzymes ejecting or capturing protons at the membrane surface. We consider these data in their relation to the mechanism of energy conversion in the living cell.
Mechanism of Primary Proton Transfer in Bacteriorhodopsin
Structure, 2004
The net effect is the transfer of one proton from the IWR cytoplasmic to the extracellular side of the membrane Heidelberg University (recently reviewed in Subramaniam et al., 1999; Lanyi, Im Neuenheimer Feld 368 2000; Neutze et al., 2002). The resulting electrochemical Heidelberg gradient is used by the cell to synthesize adenosine Germany triphosphate (ATP). The first proton transfer occurs be-2 Molecular Biophysics Department tween the L and M states on a time scale of 01ف s German Cancer Research Center (Ludman et al., 1998). In this key step, a proton is trans-Im Neuenheimer Feld 580 ferred from the retinal Schiff base to Asp85. The mecha-D-69120 Heidelberg nism of this primary step is still under much debate Germany (Lanyi 2000; Neutze et al., 2002). Available crystal struc-3 Theoretische Physik tures suggest different orientations of the Schiff base University of Paderborn NH group (Figure 1B), leading to controversy as to how Warburger Strasse 100 13-cis retinal is twisted before deprotonation. This is 33098 Paderborn essential because it determines the transfer mechanism, Germany and also because it has been proposed that retinal untwisting upon deprotonation triggers the conformational changes required by subsequent vectorial transport (Lanyi and Schobert, 2003). Another question is how can Summary the proton be transferred over the 4ف Å distance, which is too long for a direct jump. Various alternatives have Recent structures of putative intermediates in the been proposed, involving proton wires via intermediate bacteriorhodopsin photocycle have provided valuable proton carriers, either on the Thr89 (Subramaniam and snapshots of the mechanism by which protons are Henderson, 2000; Edman et al., 2003) or on the Asp212 pumped across the membrane. However, key steps (Lanyi and Schobert, 2003) side of the retinal (Figure remain highly controversial, particularly the proton 1C). A proton wire allows transfer over larger distances transfer occurring immediately after retinal trans→cis because the proton given by the Schiff base is not the photoisomerization. The gradual release of stored ensame proton accepted by Asp85. This is illustrated as ergy is inherently nonequilibrium: which photocycle follows for the case of a single intermediate carrier (I): intermediates are populated depends not only on their energy but also on their interconversion rates. To un-SB-H1 … I-H2 … Asp85 → SB … H1-I … H2-Asp85. derstand why the photocycle follows a productive (i.e., pumping), rather than some unproductive, relaxation The need to use theoretical methods to address these pathway, it is necessary to know the relative energy intertwined questions has been stressed again recently barriers of individual steps. To discriminate between (Edman et al., 2003). Indeed, the gradual release of the many proposed scenarios of this process, we comstored energy to drive vectorial proton transfer is inherputed all its possible minimum-energy paths. This reently nonequilibrium. Although there exist numerous veals that not one, but three very different pathways high-resolution X-ray crystallographic and electron mihave energy barriers consistent with experiment. This croscopic structures of bacteriorhodopsin and its putaresult reconciles the conflicting views held on the tive photocycle intermediates (Belrhali et al., 1999; mechanism and suggests a strategy by which the pro-Luecke et al., 1999; Edman et al., 1999; Royant et al., tein renders this essential step resilient. and assigning the relevant intermediates Introduction is difficult. From the simulation point of view, this means that transient states are populated not only based on Bacteriorhodopsin is a seven-helical light-driven proton
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.