Proton transport by bacteriorhodopsin through an interface film

Hwang, San-Bao; Korenbrot, Juan I.; Stoeckenius, Walther

doi:10.1007/bf01868148

Cited by 64 publications

(25 citation statements)

References 34 publications

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“…The 5-to 10-nm red shift of the action spectrum with respect to the absorption spectrum is within the bandwidth of the interference filters used; however, the shift may also reflect contributions from the excitation of red-shifted intermediates in the photocycle. Very similar action spectra have been reported for purple membrane based on the wavelength dependence of tAbsorption spectra were recorded in 1% DMPC/1% CHAPS/0.2% SDS in 10 mM phosphate buffer at pH 6. photovoltages in oriented films (11,12). Fig.…”

Section: Resultsmentioning

confidence: 99%

Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.

Subramaniam

Marti

Khorana

1990

Proc. Natl. Acad. Sci. U.S.A.

177

127

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Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85 --Glu and Arg-82 -* Ala mutants exist in a pH-dependent equilibrium between purple (A..-550/540 nm) and blue (A..=x= 600/590 nm) forms of the pigment.Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85 -* Asn mutant displays a blue chromophore (A.,. = 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form.In contrast, the Asp-212 -+ Asn mutant is purple (A.. 1 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (a) the pKa of the purple-to-blue transition is directly influenced by the pKa of the carboxylate at residue 85 and (it) the relative strengths of interaction between the protonated Schiff base, Asp-85, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.Structure-function studies with bacteriorhodopsin (bR) have shown that replacement of the membrane-buried residues has profound effects on steady-state proton pumping in reconstituted vesicles (1). Further insights into the specific role of the above aspartic residues in proton translocation have come from studies of the photocycles of bR mutants with the techniques of low-temperature and time-resolved spectroscopy (2-4). Measurements of lightinduced conductivity changes in mixed micelles have demonstrated that the Asp-96 -> Asn (D96N) mutation slows down the kinetics of proton uptake by about two orders of magnitude, without a significant effect on either the kinetics or the quantum yield of proton release (3). Optical spectroscopic experiments and photovoltage measurements with this mutant show that the rate of decay of the M intermediate of the photocycle and the associated charge movement are greatly slowed down (4, 5). These results show clearly that Asp-96 plays a key role in the proton uptake step of the photocycle. Similar studies with the mutants D85E and D85N have indicated a role for Asp-85 in proton release. Thus, replacement of Asp-85 with Asn results in complete loss of proton transport activity (1), and optical spectroscopic measurements detect no significant formation of an M-like intermediate except at high pH (6, 7). The D85E mutant was shown to be partially active in proton translocation, with increased formation of an M-like photointermediate at higher pH values (7). Fourier-transform IR spectroscopy experiments suggest that Asp-85 serves as a proton acceptor in the early phase of the photocycle (2). In related studies, slower photocycles and lower quantum yields have been observed for mutants in which Asp-115 and Asp-212 are ...

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Section: Resultsmentioning

confidence: 99%

Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.

Subramaniam

Marti

Khorana

1990

Proc. Natl. Acad. Sci. U.S.A.

177

127

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“…The ratio of the steady-state current to the peak current (n-decane) is increased by a factor of 100-1000 compared with the sandwich-like structure (9). It should be noted that the dark current is 10 as in a except that n-hexadecane was used instead of n-decane. experiment showed that the sign of the photocurrent seems to be distributed randomly.…”

Section: Methodsmentioning

confidence: 99%

“…Other methods for studying the photoelectric effect of bacteriorhodopsin have been reported. Purple membrane was transferred to an air/water or oil/water interface (10). Another approach consisted of combining purple membrane vesicles with lipid impregnated membrane filters (11,12).…”

mentioning

confidence: 99%

Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.

Bamberg¹,

Dencher²,

Fahr³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Various methods to incorporate bacteriorhodopsin in black lipid membranes are reported. Both purple membrane patches and monomeric bacteriorhodopsin were used as starting material. The incorporation ofbacteriorhodopsin into planar lipid bilayers was achieved by the following methods. (i) Purple membrane patches were transferred from water to solutions of lipids in n-alkanes. Black membranes were formed from such-organic suspensions.(ii) Lipid layers containing solvent and purple membranes were spread on an air/water interface. These layers were used to form planar bilayers. (iii) Vesicles containing purple membranes or monomeric bacteriorhodopsin were spread on an air/ water interface and, from the resulting layer, bilayers were formed. On illumination, steady-state photocurrents were observed in all three cases, indicating that these methods lead to functional transmembraneous integration of the .protein in the planar black lipid membrane. The influence ofan applied electric field on the pumping process was studied on membranes formed by using method i. At co2o. mV, the photocurrent tends to zero. Furthermore, it was. possible to make planar lipid bilayers pho, toelectrically active by adding vesicles containing monomeric bacteriorhodopsin to the bathing solution. Because, in this case, only transient photocurrents were observed, it can be concluded that the vesicles are attached to but not fused with the black lipid membrane.

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“…The method is based on the concept of spreading, not the isolated protein, but rather a mixture of purple membrane fragments and lipid. We present here an analysis of the physical and spectroscopic properties of these interface films and, in the following paper (Hwang, Korenbrot & Stoeckenius, 1977), a study of the function of bacteriorhodopsin in the films.…”

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confidence: 99%

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

1977

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A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.

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Proton transport by bacteriorhodopsin through an interface film

Cited by 64 publications

References 34 publications

Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.

Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.

Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

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