Purple membrane vesicles: Morphology and proton translocation

Hwang, San-Bao; Stoeckenius, Walther

doi:10.1007/bf01869523

Cited by 97 publications

(59 citation statements)

References 35 publications

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“…After three extractions, 25% of the initial lipid phosphorus remained in the membrane and thin-layer chromatography of the total lipid showed proportional decrease of all of the main lipid components. The action of CHAPS on pm is similar to that of cholate/deoxycholate (30) in the extent of lipid removal and the absorption shift. In the case of cholate/deoxycholate, it is known that a lattice is still present after lipid removal (6,30), and the presence of an exciton band in the CD spectra (not shown) indicates that the aggregated state is also preserved after CHAPS delipidation.…”

Section: Resultsmentioning

confidence: 74%

“…The action of CHAPS on pm is similar to that of cholate/deoxycholate (30) in the extent of lipid removal and the absorption shift. In the case of cholate/deoxycholate, it is known that a lattice is still present after lipid removal (6,30), and the presence of an exciton band in the CD spectra (not shown) indicates that the aggregated state is also preserved after CHAPS delipidation. CHAPS has advantages over cholate/deoxycholate because its use is not restricted to alkaline pH where the protein stability is reduced, and residual detergent will not contribute to the surface charge of the membrane over a wide pH range.…”

Section: Resultsmentioning

confidence: 74%

“…The small absorbance change observed upon delipidation, which is similar to that seen with other detergents or lipid replacement, where the function of bR is apparently not substantially impaired (e.g., refs. 30,32,33), argues that the structure of the molecule is not altered significantly by delipidation. This is borne out by structural studies on partially delipidated membranes (6).…”

Section: Discussionmentioning

confidence: 99%

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Effect of lipid surface charges on the purple-to-blue transition of bacteriorhodopsin.

Szundi¹,

Stoeckenius²

1987

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

116

104

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Purple membrane (ma.. = 568 nm) can be converted to blue membrane (kma. = 605 nm) by either acid titration or deionization. Partially delipidated purple membrane, containing only 25% of the initial lipid phosphorus, could be converted to a blue form by acid titration but not by deionization. This reversible transition of delipidated mem, brane did not require the presence of other cations, and the pK of the color change that in native membrane under similar conditions is between 3.0 and 4.0 was shifted to 1.4. We conclude that the purple-to-blue transition is controlled by proton concentration only and that, in native membranes, the cations act only by raising the low surface pH generated by the acidic groups of the lipids. The observation that extraction of lipids from deionized native membrane converts its color from blue to purple further confirms this conclusion. The two states of the membrane probably reflect two preferred conformations of bacteriorhodopsin, which are controlled by protonation changes at the surface of the membrane and differ slightly in the spatial distribution of charges around the chromophore.The retinylidene protein bacteriorhodopsin (bR) functions as a light-driven proton pump (1, 2). It is the only protein in the purple membrane (pm) and is arranged in a hexagonal lattice (3, 4). Purple membrane contains a variety of diether lipids, amounting to about 25% by weight, that fill the spaces between bR molecules in the lattice and are all in close contact with the protein (3-6). Most of the lipids are acidic (80%); 70% are phospholipids, mostly the diether analogue of phosphatidylglycerophosphate, and 30% are glycosulfolipids (7,8).Acidification or removal of cations from pm suspensions shifts the absorption maximum of bR from 568 to 605 nm (1, 9-15). The chromophores in these acid or deionized blue membranes are indistinguishable in absorption and resonance Raman spectra (16). The color of bR is apparently controlled by the distribution of charges in the chromophore, specifically protonation and a charge pair or dipole near the P-ionone ring that cause a large red shift of the retinal absorbance; a counterion-i.e., a negatively charged amino acid residue near the protonated Schiff base-stabilizes it with a concomitant reduction of the red shift (17-19). Protonation of this counterion has been suggested as the cause for the purple-to-blue transition. The purple color returns upon further acidification and formation of this "acid purple" chromophore has been attributed to restoration of the negative charge by binding of an anion at the site protonated in the purple-to-blue transition (11) or to protonation of a second negatively charged group (12).The original purple color of the membrane can be restored by addition of salt and alkalinization. Binding of three to five cations to specific sites is generally believed to be required for the color change (13,14,20,21) and the binding sites with the highest affinity may not affect the color change (21). Red shifts of the bR absorption ba...

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Section: Resultsmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Effect of lipid surface charges on the purple-to-blue transition of bacteriorhodopsin.

Szundi¹,

Stoeckenius²

1987

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

116

104

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“…The activity was first reconstituted by Racker and Stoeckenius (8,9) without removal of the endogenous phospholipids of the purple membrane. Subsequently, a number of studies on solubilization of the purple membrane and exchange or removal of the endogenous phospholipids have been reported (10)(11)(12)(13)(14)(15). However, complete delipidation and satisfactory reconstitution of the proton pumping activity have so far not been achieved.…”

mentioning

confidence: 99%

Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid.

Huang¹,

Bayley²,

Khorana³

1980

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

102

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Solubilizations of the purple membrane from Halobacterium halobium with the detergent Tritain X-100 followed by gel filtration in deoxycholate solution gave bacteriorhodopsin that was more than 99% free from endogenous lipid. The delipidated bacteriorhodopsin was reconstituted with exogenous phospholipids to form vesicles which on illumination efficiently translocated protons. The direction of proton pumping was from the outside to the interior of the vesicles, indicating that the orientation of bacteriorhodopsin in the vesicles was opposite to that in the bacterial membrane. This orientation was confirmed by cleavage of the carboxyl terminus of the protein by proteolysis from the outside of the vesicles.

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“…29,30 The native BR was solubilized by 10% OG for 2 h at 4 C, fractionated by sucrose density gradient centrifugation (20-60 w/w %) at 120,000g for 20 h, and reconstituted into liposomes by dialysis. 31 The photochemical properties of the native BR reconstituted into egg PC (Fig. 4 black trace) were essentially identical to those of the cell-free product (red trace 29,32 In these measurements, the samples were diluted 1/5 with buffer (50 mM Tris-HCl and 400 mM NaCl).…”

Section: Functional Assay Of Proteoliposomesmentioning

confidence: 73%

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

et al. 2009

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Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane a-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

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Purple membrane vesicles: Morphology and proton translocation

Cited by 97 publications

References 35 publications

Effect of lipid surface charges on the purple-to-blue transition of bacteriorhodopsin.

Effect of lipid surface charges on the purple-to-blue transition of bacteriorhodopsin.

Delipidation of bacteriorhodopsin and reconstitution with exogenous phospholipid.

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

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