Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding

Zubov, B. V.; Tsuji, Kinko; Hess, Benno

doi:10.1016/0014-5793(86)80543-9

Cited by 17 publications

(16 citation statements)

References 6 publications

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“…Time resolved titrations of specific residues are generally difficult to perform in proteins (for a recent review see [25]). However, due to the associated changes in color, they have been carried out in bR using conventional stopped‐flow techniques for both the Schiff base [26–28]and Asp‐85 [12, 27–31]. A characteristically unclear feature of the Asp‐85 titration has been its multiphasic kinetic nature, ranging from tens of milliseconds to hours [3, 12, 28, 29, 31].…”

Section: Resultsmentioning

confidence: 99%

“…However, due to the associated changes in color, they have been carried out in bR using conventional stopped‐flow techniques for both the Schiff base [26–28]and Asp‐85 [12, 27–31]. A characteristically unclear feature of the Asp‐85 titration has been its multiphasic kinetic nature, ranging from tens of milliseconds to hours [3, 12, 28, 29, 31]. In a recent work [32], we proposed a kinetic model for the cation‐induced blue↔purple transition in which the rate‐determining step of the Asp‐85 titration is proton transfer through an appropriate channel, rather than the binding of the metal cation.…”

Section: Resultsmentioning

confidence: 99%

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Titration kinetics of Asp‐85 in bacteriorhodopsin: exclusion of the retinal pocket as the color‐controlling cation binding site

Bressler

Ottolenghi

et al. 1997

FEBS Letters

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Titration kinetics of Asp‐85 in bacteriorhodopsin: exclusion of the retinal pocket as the color‐controlling cation binding site

Bressler

Ottolenghi

et al. 1997

FEBS Letters

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“…The major part of the absorbance change required >10 min in 20 mM CHAPS and even longer times at concentrations much below the critical micelle concentration. Binding of a detergent molecule or a cation to a specific site would be a much faster process, and cations do indeed restore the purple color in less than a second (13,31).…”

Section: Resultsmentioning

confidence: 99%

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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“…(c) Different sample preparation techniques resulting in possible differences in protein conformation in the hydrated membrane stack used in x-ray work and glucose-embedded sheets used in this study. The binding characteristics of cations, particularly divalent cations, in the process of blue to purple transition have been recently investigated by optical absorption (Kimura et al, 1984;Zubov et al, 1986;Chang et al, 1986;Ariki and Lanyi, 1986) or by ESR (Du-nach et al, 1986Corcoran et al, 1987). Duniach et al (1987), find five medium and high affinity sites (with Kd = 0.6-26.0 ,M at pH 5.0 and 20-50 ,uM at pH 7.0) and five low-affinity sites (50 mM) for Mn2" on bR.…”

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confidence: 99%

High sensitivity electron diffraction analysis. A study of divalent cation binding to purple membrane

Mitra

Stroud

1990

Biophysical Journal

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A sensitive high-resolution electron diffraction assay for change in structure is described and harnessed to analyze the binding of divalent cations to the purple membrane (PM) of Halobacterium halobium. Low-dose electron diffraction patterns are subject to a matched filter algorithm (Spencer, S. A., and A. A. Kossiakoff. 1980. J. Appl. Crystallogr. 13:563-571). to extract accurate values of reflection intensities. This, coupled with a scheme to account for twinning and specimen tilt in the microscope, yields results that are sensitive enough to rapidly quantitate any structure change in PM brought about by site-directed mutagenesis to the level of less than two carbon atoms. Removal of tightly bound divalent cations (mainly Ca2+ and Mg2+) from PM causes a color change to blue and is accompanied by a severely altered photocycle of the protein bacteriohodopsin (bR), a light-driven proton pump. We characterize the structural changes that occur upon association of 3:1 divalent cation to PM, versus membranes rendered purple by addition of excess Na+. High resolution, low dose electron diffraction data obtained from glucose-embedded samples of Pb2+ and Na+ reconstituted PM preparations at room temperature identify several sites with total occupancy of 2.01 +/- 0.05 Pb2+ equivalents. The color transition as a function of ion concentration for Ca2+ or Mg2+ and Pb2+ are strictly comparable. A (Pb2(+)-Na+) PM Fourier difference map in projection was synthesized at 5 A using the averaged data from several nominally untilted patches corrected for twinning and specimen tilt. We find six major sites located on helices 7, 5, 4, 3, 2 (nomenclature of Engelman et al. 1980. Proc. Natl. Acad. Sci. USA. 77:2023-2027) in close association with bR. These partially occupied sites (0.55-0.24 Pb2+ equivalents) represent preferential sites of binding for divalent cations and complements our earlier result by x-ray diffraction (Katre et al. 1986. Biophys. J. 50:277-284).

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Transition kinetics of the conversion of blue to purple bacteriorhodopsin upon magnesium binding

Cited by 17 publications

References 6 publications

Titration kinetics of Asp‐85 in bacteriorhodopsin: exclusion of the retinal pocket as the color‐controlling cation binding site

Titration kinetics of Asp‐85 in bacteriorhodopsin: exclusion of the retinal pocket as the color‐controlling cation binding site

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

High sensitivity electron diffraction analysis. A study of divalent cation binding to purple membrane

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