Effects of Bleaching and Regeneration on the Purple Membrane Structure of Halobacterium halobium

Becher, B.; Cassim, Joseph Y.

doi:10.1016/s0006-3495(77)85588-4

Cited by 74 publications

(62 citation statements)

References 32 publications

(50 reference statements)

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“…At least part of the retinaloxime will still occupy the retinal-binding site as deduced from CD spectroscopy (23). I-V curves of such samples showed behavior similar to that of native bR in terms of current magnitude, but they did not show any response to green light as expected in samples lacking absorption in this region (Fig.…”

Section: Resultsmentioning

confidence: 63%

Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers

Jin

Friedman²,

Sheves³

et al. 2006

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

156

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Studying electron transport (ET) through proteins is hampered by achieving reproducible experimental configurations, particularly electronic contacts to the proteins. The transmembrane protein bacteriorhodopsin (bR), a natural light-activated proton pump in purple membranes of Halobacterium salinarum, is well studied for biomolecular electronics because of its sturdiness over a wide range of conditions. To date, related studies of dry bR systems focused on photovoltage generation and photoconduction with multilayers, rather than on the ET ability of bR, which is understandable because ET across 5-nm-thick, apparently insulating membranes is not obvious. Here we show that electronic current passes through bR-containing artificial lipid bilayers in solid ''electrode-bilayer-electrode'' structures and that the current through the protein is more than four orders of magnitude higher than would be estimated for direct tunneling through 5-nm, water-free peptides. We find that ET occurs only if retinal or a close analogue is present in the protein. As long as the retinal can isomerize after light absorption, there is a photo-ET effect. The contribution of light-driven proton pumping to the steady-state photocurrents is negligible. Possible implications in view of the suggested early evolutionary origin of halobacteria are noted. molecular electronics ͉ vesicles ͉ bioelectronics B acteriorhodopsin (bR) is a protein-chromophore complex that serves as a light-driven proton pump in the purple membrane (PM) of Halobacterium salinarum (1). It has been shown that the protein is composed of seven transmembrane helices with a retinal chromophore covalently bound in the central region via a protonated Schiff base to a lysine residue (Fig. 1A). The PM is organized in a 2D hexagonal crystal lattice with a unit cell dimension of Ϸ6.2 nm. Electron crystallography has indicated that bR is organized into trimers in which lipids mediate intertrimer contact (2). Light absorption by bR initiates a multistep reaction cycle with several distinct spectroscopic intermediates: J 625 , K 590 , L 550 , M 412 , N 560 , and O 640 . More details on the molecular alterations that occur during the photocycle were recently obtained from x-ray diffraction studies (see ref. 3 for a recent review). The light-adapted form of bR contains only all-trans retinal, whereas the dark-adapted form contains a 1:1 mixture of 13-cis and all-trans (4). Because of its long-term stability against thermal, chemical, and photochemical degradation and its desirable photoelectric and photochromic properties, bR has attracted much interest as a material for biooptics and bioelectronics (5). Most of these efforts focused on multilayers and their photovoltage͞photocurrent generation (6-8) and photoconduction (9).In principle, PM patches (Ϸ5 nm thick, a few m in size; see Fig. 1B) can serve as a model protein material that is important for both planar junction fabrication and current transport measurements, because the 5-nm membrane is well beyond the thickness over which tu...

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Section: Resultsmentioning

confidence: 63%

Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers

Jin

Friedman²,

Sheves³

et al. 2006

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

156

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“…In this case, slow oxidation with molecular oxygen is observed in the dark for the label at the 103 residue, which is considerably accelerated following irradiation of the retinal oxime, absorbing at 360 nm. It was previously shown that retinal oxime occupies the retinal binding site and exhibits a CD signal (38). Moreover, AFS studies 3 detected protein conformational alterations induced by light absorption by the retinal oxime.…”

Section: Discussionmentioning

confidence: 95%

Bacteriorhodpsin Experiences Light-induced Conformational Alterations in Nonisomerizable C13=C14Pigments

Aharoni¹,

Weiner²,

Ottolenghi³

et al. 2000

Journal of Biological Chemistry

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The mechanism by which bacteriorhodopsin is activated following light absorption is not completely clear. We have detected protein conformational alterations following light absorption by retinal-based chromophores in the bacteriorhodopsin binding site by monitoring the rate of reduction-oxidation reactions of covalently attached spin labels, using EPR spectroscopy. It was found that the reduction reaction with hydroxylamine is light-catalyzed in the A103C-labeled pigment but not in E74C or M163C. The reaction is light-catalyzed even when isomerization of the C 13 ‫؍‬C 14 bond of the retinal chromophore is prevented. The reverse oxidation reaction with molecular oxygen is effective only in apomembrane derived from the mutant A103C. This reaction is light-accelerated following light absorption of the retinal oxime, which occupies the binding site. The light-induced acceleration is evident also in "locked" bacteriorhodopsin in which isomerization around the C 13 ‫؍‬C 14 bond is prevented. It is evident that the chromophore-protein covalent bond is not a prerequisite for protein response. In contrast to the case of the retinal oxime, a reduced C‫؍‬N bond A103C-labeled pigment did not exhibit acceleration of the oxidation reaction following light absorption. Acceleration was observed, however, following substitution of the polyene by groups that modify the excited state charge delocalization. It is suggested that protein conformational alterations are induced by charge redistribution along the retinal polyene following light absorption. Bacteriorhodopsin (bR)1 is the integral protein of the purple membrane of Halobacterium salinarum and serves as a lightdriven proton pump (1-3). It is composed of seven transmembrane helices enclosing the binding pocket for an all-transretinal chromophore, which is bound to Lys 216 via a protonated Schiff base (SBH) ϩ . Absorption of a photon by the retinal induces an all-trans 3 13-cis isomerization, which initiates a photocycle with several distinct spectroscopic intermediates, J 625 , K 590 , L 550 , M 412 , N 560 , and O 640 . It is well established that the retinal in K 590 is characterized by a 13-cis configuration (4,5). Deprotonation of the protonated Schiff base takes place during the L to M transition, which is accompanied by protonation of Asp 85 and the appearance of a proton at the extracellular surface. The Schiff base is reprotonated during the M to N transition from the proton donor Asp 96 , which is finally reprotonated from the cytoplasmic side during the recovery initial state of bR.It is widely assumed that all light-induced protein conformational alterations in retinylidine proteins, including bR, are initiated by isomerization of the retinal chromophore. However, alternative approaches have been suggested in which isomerization is not the only trigger for biological activity or protein structural alterations (6). One approach attributed protein conformational alterations to large charge redistribution in the retinal chromophore developed following light absorp...

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“…However, only moderate success has been achieved in identifying molecular motions within the apoprotein and in correlating them with the well-characterized photochemical intermediates. The results of the research done on this problem (10)(11)(12)(13)(17)(18)(19) disagree as to the origins and the extent ofthe aromatic amino acid perturbations. Changes in the intrinsic protein fluorescence ofbR, although small, seem to offer promising opportunities to monitor the time-dependent interactions (involving tyrosine and tryptophan residues) within the chromoprotein during the photochemical cycle.…”

mentioning

confidence: 94%

“…Less is known about the involvement of the protein moiety in the photochemical cycle and related processes, although the structural-functional unit of retinal and bacterio-opsin is evident. Ultrafast spectroscopic results (3,4) suggest that the protein may respond almost instantaneously with a rapid protontransfer reaction to photon absorption by the chromophore; numerous studies (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) additionally indicate that the protein undergoes a series ofconformational changes as the photochemical process proceeds. However, only moderate success has been achieved in identifying molecular motions within the apoprotein and in correlating them with the well-characterized photochemical intermediates.…”

mentioning

confidence: 99%

Time-resolved protein fluorescence studies of intermediates in the photochemical cycle of bacteriorhodopsin.

Fukumoto¹,

Hopewell²,

Karvaly³

et al. 1981

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

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The photolysis-induced changes in the protein fluorescence intensity (at 320 nm) during the proton-pumping cycle of bacteriorhodopsin were examined by a delayed two-pulse technique in the time range 1 psec-20 msec at room temperature. No detectable change in the protein fluorescence intensity was observed on the earliest time scale within the lifetime of the intermediate K when retinal apparently undergoes the largest structural changes. The time dependence of the relative changes in fluorescence intensity did, however, display a close correlation with the population of the LM and M412 intermediates. From a computer numerical fit of the data, with available published kinetic parameters, the protein fluorescence quantum yields of the K590, L I and M412 intermediates are found to be 1.0, 0.92, and 0.80 of that for native bR570, respectively. The probable mechanisms of the observed fluorescence quenching during the photochemical cycle are qualitatively discussed.Most work done on bacteriorhodopsin (bR) has focused attention mainly on the role of the Schiff-base-linked retinal (1, 2). Less is known about the involvement of the protein moiety in the photochemical cycle and related processes, although the structural-functional unit of retinal and bacterio-opsin is evident. Ultrafast spectroscopic results (3, 4) suggest that the protein may respond almost instantaneously with a rapid protontransfer reaction to photon absorption by the chromophore; numerous studies (5-19) additionally indicate that the protein undergoes a series ofconformational changes as the photochemical process proceeds. However, only moderate success has been achieved in identifying molecular motions within the apoprotein and in correlating them with the well-characterized photochemical intermediates. The results of the research done on this problem (10-13, 17-19) disagree as to the origins and the extent ofthe aromatic amino acid perturbations. Changes in the intrinsic protein fluorescence ofbR, although small, seem to offer promising opportunities to monitor the time-dependent interactions (involving tyrosine and tryptophan residues) within the chromoprotein during the photochemical cycle. This communication reports on some correlations between fluorescence intensity changes and the populations of the photochemical intermediates in the microsecond and millisecond time domains.MATERIALS AND METHODS Carotenoid-free bR was isolated and purified by the method of Becher and Cassim (20) and used without sonication. Suspensions were prepared in doubly distilled water at a concentration of 22 AM + 10% (e = 63,000 liters/mol cm) in all experiments.Samples placed in a semimicro quartz absorption cuvette with a 1.0-cm optical pathlength (Markson, type E-18, Del Mar, CA) were light adapted prior to fluorescence measurements by exposure to standard room light for at least 2 hr. To minimize the influence of polarization effects (the possible excitation of populations with different polarization properties) we illuminated the sample through one of the ...

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Effects of Bleaching and Regeneration on the Purple Membrane Structure of Halobacterium halobium

Cited by 74 publications

References 32 publications

Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers

Bacteriorhodopsin (bR) as an electronic conduction medium: Current transport through bR-containing monolayers

Bacteriorhodpsin Experiences Light-induced Conformational Alterations in Nonisomerizable C13=C14Pigments

Time-resolved protein fluorescence studies of intermediates in the photochemical cycle of bacteriorhodopsin.

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