Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release

Balashov, Sergei P.; Imasheva, Eleonora S.; Govindjee, Rajni; Ebrey, Thomas G.

doi:10.1016/s0006-3495(96)79591-7

Cited by 218 publications

(356 citation statements)

References 52 publications

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“…Moreover, the experimental pH-dependence of the purple-to-blue transition is complex, apparently involving couplings to elements of the extracellular proton-release system. 39,40 The photocycle is known to occur via the same stages 41 in an extremely wide range of pH, from at least 5 to 9, which is larger than the typical uncertainties associated with pK a predictions based on the methodologies 32,34 used here. The present work focuses on relative changes of proton affinities during the cycle, which are less sensitive to methodological uncertainties.…”

Section: Br (Equilibrium) Statementioning

confidence: 77%

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

Onufriev

Smondyrev

Bashford

2003

Journal of Molecular Biology

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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 individualgroup pK a values traditionally used. When combined with state-to-state transition rules based on accessibility considerations, a model for nonequilibrium proton flow is obtained. Such methods should also be applicable to other active proton-transport systems.

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Section: Br (Equilibrium) Statementioning

confidence: 77%

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

Onufriev

Smondyrev

Bashford

2003

Journal of Molecular Biology

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“…In the dark adapted state, the chromophore is in an equilibrium between the all trans (34%) and 13-cis (66%) configurations (34). In wt-bR the pH dependence of the dark adaptation rate and the fraction of molecules in the blue state were shown to be the same (12). We have measured the dark adaptation rate for wt, R82A, G231C, and R82A/G231C at selected pH values (data not shown).…”

Section: Characterization Of the Unphotolyzed Single And Double Mutanmentioning

confidence: 95%

“…The kinetics of light-dark adaptation of the chromophore in the unphotolyzed state of bR is also controlled by the protonation state of Asp-85 (12). In the light-adapted state the retinylidene chromophore is 100% in the all trans conformation.…”

Section: Characterization Of the Unphotolyzed Single And Double Mutanmentioning

confidence: 99%

“…Arg-82 is located one helix turn away from Asp-85, facing toward the proton channel (9,10). Steadystate and time-resolved UV/Vis absorption (11)(12)(13) and Fourier transform infrared (14) spectroscopy results indicate that Asp-85, Glu-204, , and Arg-82 interact via a direct or indirect coupling of their pK values. Proton release to the extracellular medium may be facilitated by a hydrogen-bonded network between these residues (10,13,15,16).…”

mentioning

confidence: 98%

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Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Alexiev¹,

Mollaaghababa²,

Khorana³

et al. 2000

Journal of Biological Chemistry

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Evidence is presented for long range interactions between the extracellular and cytoplasmic parts of the heptahelical membrane protein bacteriorhodopsin in the mutant R82A and its second site revertant R82A/ G231C. (i) In the double mutants R82A/G72C and R82A/ A160C, with the cysteine mutation on the extracellular or cytoplasmic surface, respectively, the photocycle is the same as in the single mutant R82A with an accelerated deprotonation of the Schiff base and a reversed order of proton release and uptake. Proton release and uptake kinetics were measured directly at either surface by using the unique cysteine residue as attachment site for the pH indicator fluorescein. Whereas in wild type proton uptake on the cytoplasmic surface occurs during the M-decay ( ϳ 8 ms), in R82A it occurs already during the first phase of the M-rise ( < 1 s). (ii) The introduction of a second mutation at the cytoplasmic surface in position 231 (helix G) restores wild type ground state absorption properties, kinetics of photocycle and of proton release, and uptake in the mutant R82A/G231C. In addition, kinetic H/D isotope effects provide evidence that the proton release mechanism in R82A/G231C and in wild type is similar. These results suggest the existence of long range interactions between the cytoplasmic and extracellular surface domains of bacteriorhodopsin mediated by salt bridges and hydrogen-bonded networks between helices C (Arg-82) and G (Asp-212 and Gly-231). Such long range interactions are expected to be of functional significance for activation and signal transduction in heptahelical Gprotein-coupled receptors.Allosteric interactions are well known and of great importance for water soluble enzymes, where ligand or substrate binding to a specific site alters the conformation and changes the affinity at a different site of the protein (for reviews see Refs. 1 and 2). For membrane-bound G-protein-coupled receptors analogous effects are expected to be of considerable functional significance. For this class of proteins ligand binding occurs mostly on the extracellular surface resulting in the active form of the receptor with binding and activation of the G-protein at the opposite cytoplasmic surface of the protein.The membrane protein bacteriorhodopsin (bR) 1 shares the heptahelical bundle motif with G-protein-coupled receptors. In this report, we present evidence for long range interactions between the extracellular and cytoplasmic parts of bR based on evidence from the mutant R82A and its second site revertant R82A/G231C.Bacteriorhodopsin acts as a light-driven proton pump in the plasma membrane of the archaebacterium Halobacterium salinarium. A retinylidene chromophore is bound via a protonated Schiff base linkage to Lys-216. Upon flash excitation bR undergoes a cyclic photoreaction with distinct spectroscopic intermediates and proton translocation from one side of the membrane to the other (for reviews see Refs. 3-5). In the first half of this photocycle, the Schiff base becomes deprotonated during the transition to the ...

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“…Isomerization seems to be catalyzed by the protonated form of Asp-85 which, in turn, is protonated by Glu-204. It is known that protonation of Asp-85 is required for cis-trans isomerization during dark adaptation [32,33]. If Glu-204 is deprotonated in the alkaline bR form ground state, then the L intermediate appearing during the alkaline bR photocycle should have deprotonated Asp-85 and Glu-204 just as the L intermediate formed from neutral bR at pH higher than the pK of Glu-204 in the excited state (pK=5.4).…”

Section: Resultsmentioning

confidence: 99%

Complicated character of the M decay pH dependence in the D96N mutant is due to the two pathways of the M conversion

1996

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~bstract At high ionic strength, the pH dependence of the M intermediate decay in a photocycle of the D96N mutant bacteriorhodopsin shows a complicated behavior which is found lo be due to the coexistence of two pathways of the M conversion. The M decay which dominates at pH < 5 is coupled to the proton uptake from the cytoplasmic surface and proceeds probably through the N intermediate. This pathway is inhibited by glutaraldehyde, the potent inhibitor of M decay in the wildtype bacteriorhodopsin and of the azide-facilitated M decay in the D96N mutant. Another pathway of the M decay is predominant at pH > 5. This pathway is insensitive to glutaraldehyde and some other similar inhibitors (lutetium ions, sucrose and glycerol). On the other hand, it is sensitive to the pK changes ,ff the group X (Glu-204) in the outward proton pathway. Possibly, the M decay through this pathway represents a reverse H + transport process (the proton uptake from the external surface) and proceeds via the L intermediate.

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Titration of aspartate-85 in bacteriorhodopsin: what it says about chromophore isomerization and proton release

Cited by 218 publications

References 52 publications

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

Proton Affinity Changes Driving Unidirectional Proton Transport in the Bacteriorhodopsin Photocycle

Evidence for Long Range Allosteric Interactions between the Extracellular and Cytoplasmic Parts of Bacteriorhodopsin from the Mutant R82A and Its Second Site Revertant R82A/G231C

Complicated character of the M decay pH dependence in the D96N mutant is due to the two pathways of the M conversion

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