A photosensitive product of sodium borohydride reduction of bacteriorhodopsin

Peters, Jutta; Peters, Reiner; Stoeckenius, Walther

doi:10.1016/0014-5793(76)81019-8

Cited by 51 publications

(34 citation statements)

References 33 publications

(9 reference statements)

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“…The procedure of Peters et al (19) was used, but the concentration of PM in the mixtures was increased in most cases so that less NaBH4 was required.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Site of attachment of retinal in bacteriorhodopsin.

Bayley¹,

Huang²,

Radhakrishnan³

et al. 1981

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

153

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The identity of the lysine residue in bacteriorhodopsin to which the chromophore, retinal, is attached as a Schiff's base has been reinvestigated. Retinal is now shown to be linked to lysine-216 and not lysine-41, as had been concluded previously. The retinal in purple membrane was replaced by [15-H]retinal, the Schiff's base linkage was reduced with NaBH4 while the sample was illuminated, and the resulting [retinyl-3H]bacterio-opsin was cleaved with CNBr. The radioactivity was present exclusively in the COOH-terminal pestide (amino acids 210-248). Sequence analysis showed that the [ H]retinal was attached to lysine-216.The same site was labeled when purple membrane was reduced with NaBH4 in the light at pH >10 or at pH 8 and when membranes modified with ether or solubilized with hexadecyltrimethylammonium bromide were reduced in the dark. Our finding places the Schiff's base linkage close to the midpoint ofthe putative membrane-spanning ae-helix that is directly connected to the COOH terminus of bacteriorhodopsin.Bacteriorhodopsin, the only protein in the purple membrane (PM) of Halobacterium halobium, catalyzes the light-driven vectorial translocation of protons and thus generates a transmembrane electrochemical gradient (1). The protein consists of a single polypeptide chain of 248 amino acids, whose amino acid sequence is now known (2, 3). A three-dimensional model of bacteriorhodopsin has been developed (4) that is compatible with the amino acid sequence, diffraction data (5, 6), and the susceptibility ofcertain regions ofthe polypeptide to proteolysis (7,8): the polypeptide is believed to traverse the membrane seven times in the form of a-helical rods.For further structural and mechanistic studies, it is important to know the site ofattachment ofretinal to the polypeptide. The chromophore is attached as a Schiff's base to the e-amino group of a lysine residue (9, 10), previously concluded to be lysine-41 (2, 3, 11) based on the results of Bridgen and Walker (12). We have reinvestigated the site of attachment; we find that retinal is attached to lysine-216. We compare our finding with the result of a recent study in which the site of attachment of retinal to bovine rhodopsin was determined (13 [Retinal-3H]PM (30 1d; 13.8 mg ofprotein ml-') or unlabeled PM at the same concentration and NaBH4 (22 mg ml-l) or NaB3H4 (25 mCi; 434 mCi mmol-1) in 50 mM Na2COJHCl, pH 10.0 (100 ul) were mixed at 00C and illuminated with a Kodak model 800 slide projector equipped with a Schott OG530 filter. The mixture was bleached within 10-15 min, and the reaction was then quenched by transferring the membranes to 100 mM sodium phosphate, pH 6.5 (1.3 ml). After 1 hr at 00C and 1 hr at 250C, the product was dialyzed against the same buffer for 1 day at 250C and then further washed by dialysis or by repeated centrifugation and suspension. Unlabeled reduced PM was produced by using the same procedure on a larger scale. An illuminated sample of [retinal-3H]PM (10 1.l; 13.8 mg ml-') was also completely reduced at pH 8 and 0C i...

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“…The procedure of Peters et al (19) was used, but the concentration of PM in the mixtures was increased in most cases so that less NaBH4 was required.…”

Section: Methodsmentioning

confidence: 99%

“…RESULTS Analysis of the Chymotryptic Fragments, C-1 and C-2: Retinal Is Attached to C-I (Residues 72-248). PM was treated with NaBH4 at pH 10.0 under illumination to fix-the retinal molecule at its binding site by reduction of the Schiffs base linkage (9,10,19). The membrane was then treated with chymotrypsin ( Fig.…”

Section: Methodsmentioning

confidence: 99%

Site of attachment of retinal in bacteriorhodopsin.

Bayley¹,

Huang²,

Radhakrishnan³

et al. 1981

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

153

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“…Purified purple membrane fragments were reduced with 1% NaBH4 at 0°C under illumination with visible light (520-600 nm) from a projector [9]. The reduced product, bRred, was washed and resuspended in 50 mM borate (pH 8.9).…”

Section: Methodsmentioning

confidence: 99%

Immobility of the chromophore in bacteriorhodopsin

et al. 1981

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“…It is generally thought that there is no cis-trans isomerization during the light-adapted cycle; however, there has been some speculation that the retinal may be retro all-trans (7) in the bM412 intermediate because of the similarity of the structure in its absorption spectrum to the absorption spectra of several known retro compounds (7,31). The isomeric configuration of retinal in various intermediates of bacteriorhodopsin has been determined by trapping the species, extracting the retinal from the protein and analyzing it (29,30).…”

mentioning

confidence: 99%

Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

Terner¹,

Campion²,

El‐Sayed³

1977

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

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A simple technique is described that uses a continuous wave laser with electromechanical modulation to obtain time-resolved Raman spectra of transient species on the millisecond timescale. The time behavior of the vibrational bands of the intermediates involved in the proton pumping of bacteriorhodopsin is determined. From these results, along with resonance enhancement and power dependence studies, the bands that appear in the continuous wave Raman spectrum of bacteriorhodopsin can be assigned to three intermediates in the photochemical cycle of bacteriorhodopsin, bR570, bL550, and bM412. The Raman spectra of bR570 and bM412 are compared with published spectra of model Schiff bases of all-trans and 13-cis retinal.Several papers have described the use of time-resolved Raman spectroscopy to obtain vibrational and kinetic information about transient species. Resonance Raman spectra have been obtained of the short-lived free radical p-terphenyl anion (1) and FeCO3 in pulsed magnetic fields (2). Intermediates of bacteriorhodopsin have been studied on the microsecond (3, 4) and nanosecond (5) timescales. In this paper we describe work on bacteriorhodopsin in the millisecond range.Bacteriorhodopsin is found in purple patches of the cell membrane of various species of halophilic bacteria. Discovered in 1971 by Oesterhelt and Stoeckenius (6), it is the only other photosynthetic system, besides the chlorophylls, known to exist in nature. Bacteriorhodopsin is structurally similar to the visual pigment rhodopsin but its biological role is quite different. Under illumination, bacteriorhodopsin cycles through several intermediates ( Fig. 1) 100-200 times per sec. The net result of this photochemical cycle is to pump protons across the cell membrane, which sets up an electrochemical gradient that is thought to drive the synthesis of ATP (7).Resonance Raman spectroscopy has been used to probe the structure of bacteriorhodopsin (8-11), rhodopsin (12-17), and the chromophore of both species, isomers of retinal (18)(19)(20)21 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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A photosensitive product of sodium borohydride reduction of bacteriorhodopsin

Cited by 51 publications

References 33 publications

Site of attachment of retinal in bacteriorhodopsin.

Site of attachment of retinal in bacteriorhodopsin.

Immobility of the chromophore in bacteriorhodopsin

Time-resolved resonance Raman spectroscopy of bacteriorhodopsin on the millisecond timescale.

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