ARCHIVES

Vol.

OF BIOCHEMISTRY

284, No. 1, January,

AND

BIOPHYSICS

pp. 1-8,

1991

Influence of the Surface Potential Membrane Structure and Activity Marie-Emmanuel

Riviere,’

Bernard

Arrio,

Robert

on the Purple Pansu,*

and Jean Faure*

C.N.R.S. URA 1116, Bio&erg&ique Membranaire, B&. 433; and T.N.R.S. des Rayonnements, B&t. 350; Universite’ de Paris-Sud, 91405 Orsay, France

Received

January

16, 1990, and in revised

form

August

16, 1990

The role of the divalent cations in the purple membrane is generally understood as the release mechanism of the blue form appearance. The reconstitution by cation addition leads to the recovery of the initial spectral properties. Numerous data are available in the literature on this matter but they are scattered, so that synthetic understanding is not easy. The role of divalent cations was studied through spectrophotometric titrations and electrophoretic mobility measurements, i.e., { potential valuations. Thus, correlations between the bacteriorhodopsin (bR) state and the whole membrane in equilibrium with a definite medium could be made. Deionization was not a fully reversible process. The absence of cations affect neither the rate of the M4i2 formation nor its lifetime but the yield of M&bR was 50% lower. The number of protons involved in the blue to purple transition of both membranes was different and the reconstitution did not erase this difference. It was observed that the number of protons dissociated upon cation addition corresponded approximately to the number of positive charges removed by deionization. Electrophoretic mobility titrations showed large differences between the membranes, illustrating the influence of the surface charge density on the pK of the transition. Taking advantage of the reversible light adaptation process, the reciprocal influence of the charge density of the membrane surface and the retinal state in bR was shown. Specificity of the divalent cations was questioned by a direct substitution of them by imidazol, which left the membrane intact. The partial reversibility of the deionization, the decrease of the Md12 yield, the differences in the titratable protons, and the nonstrict specificity toward divalent cations suggested that another unknown factor could be removed from the membrane. 0 1991 Academic Press, Inc.

r To whom correspondence should be addressed inergetique Membranaire, Institut de Biochimie, de Paris-Sud, 91405 Orsay Cedex, France. 0003-9861/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

Inc. reserved.

URA 75, Physicochimie

at URA 1116, BioBat. 433, Universite

Bacteriorhodopsin (bR)2 is the only protein present in the purple membrane patches (PM) of Halobacterium halobium. This protein binds a retinal moiety through a Schiff base with the lysine 216 residue. The trimers of bR, with specific polar lipids, form an hexagonal lattice in the plane of the membrane. Bacteriorhodopsin functions as a light driven proton pump: when illuminated, the protein undergoes a well-characterized photocycle (1) accompanied by the vectorial proton transport (2). It has been recently shown that PM contains three or four divalent cations/bR (3, 4) which are essential for the stabilization of the purple form toward pH variations. The removal of these cations causes the spectral transition from purple to blue. The blue form is characterized by an absorbance maximum at 602 nm, a low divalent cations content, less than O.l/bR, a disorganized bR lattice (5), and a lower stability (6-8). This last characteristic is illustrated by a higher sensitivity toward bleaching agents (bright light, electric fields, sonication). The main disorder is the absence of the Md12intermediate during the photocycle of bR in the blue membrane (9). The bound divalent cations are also assumedto monitor the pK of the color-controlling residues of bR via the modification of the surface potential (10-13). Addition of cations to the blue form restores the purple color immediately; some reports also describe the color recovery by addition of small hydrophobic-charged or neutralmolecules to the blue membrane (12, 14-16). However, this reversibility is much debated since data from microcalorimetry (6), electrooptical properties (8), and reconstitution from deionized or deionized and bleached mem’ Abbreviations used: bR, bacteriorhodopsin; PM, native purple membrane; BM, native membrane in the blue form (acidified membrane); dePM, deionized membrane in the purple form; deBM, deionized membrane in the blue form; rePM, deionized membrane reconstituted by cation addition, in the purple form; rn2, halflife; nH+, number of protons implied in the spectral transition; bR,,, bR present in the purple form of the membrane (PM); bh2, bR present in the blue form of the membrane (BM). 1

2

RIVI&RE

ET

AL.

branes (7) point out the differences between the original and the cation-depleted membranes. To emphasize the relationship between the cation content and the structure of normal and depleted membranes, we studied pH and cation titrations on the retinal absorbance, which is a local indicator of the protein conformation, and on the surface charge density. These two parameters are sensitive to the whole surface modifications and consequently to the ionic equilibrium changes. An application of this relation between the chromophore environment and the surface was evidenced by the light adaptation process. Formation rates, lifetimes, and yields of the Mdlz intermediate were examined, particularly on the depleted membranes at neutral pH. From these titrations, the partial reversibility of the cation depletion was illustrated and the consequences on the Md12 yield were revealed.

mostated. The M,,, and bRETO rate constants were estimated as the average of 10 flashes on three samples. The Mb12 maximal absorbances were estimated for various laser intensities. The absorbance at 412 nm reflects the quantity of M,12 formed after the flash, i.e., the number of bR molecules that have undergone a photocycle. The maximal absorbance was reached for a laser intensity of 30 mJ and remained constant until 120 mJ. Thus, all the experiments were run with 90 mJ intensity to ensure maximal activation of the bR without any photobleaching of the sample. In addition, under these conditions, the maximal absorbance at 412 nm varied linearly with the bR concentration in the sample. The Md12 concentration was evaluated by extrapolation of its absorbance at t = 0 (e412 = 23,000 M-‘cm-’ (19)).

MATERIALS

RESULTS

AND

METHODS

Electrophoretic

Mobility

Measurements

A laser Doppler velocimetry (LDV) technique was used to determine the

Influence of the surface potential on the purple membrane structure and activity.

The role of the divalent cations in the purple membrane is generally understood as the release mechanism of the blue form appearance. The reconstituti...
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