“…This corresponds to results reported by other authors (26,27). Solubilized bacteriorhodopsin was first obtained by treatment with Triton X-100 (2% w/v) under magnetic stirring at room temperature for 20 h and afterward separated from the membrane debris by centrifugation (2 h, 100,000g).…”
Section: Specific Detergent Binding To Membrane Proteinssupporting
“…This corresponds to results reported by other authors (26,27). Solubilized bacteriorhodopsin was first obtained by treatment with Triton X-100 (2% w/v) under magnetic stirring at room temperature for 20 h and afterward separated from the membrane debris by centrifugation (2 h, 100,000g).…”
Section: Specific Detergent Binding To Membrane Proteinssupporting
“…Since its discovery in 1971, bacteriorhodopsin (BR; see Figure ) has become one of the most extensively studied of all proteins due to its ready availability, its thermal stability − and photostability, − its similarity to vertebrate visual pigments ,, and other G protein-coupled receptors, and its potential applications in photonic devices. , BR, which imparts color to the “purple membrane” (PM) of the archaebacterium Halobacterium salinarium , ,,, is a 248-residue, 26 kDa chromophoric transmembrane protein consisting of seven α-helices oriented around a common center, in which an all- trans -retinyl protonated Schiff base chromophore (ATRPSB, see Figure ) is covalently linked to the protein backbone at the Lys-216 residue. , Upon absorbing a photon of visible light, ATRPSB isomerizes to the 13- cis conformation, leading to a series of global conformational changes in BR which result in the pumping of a proton across the PM, generating a transmembrane proton gradient which is coupled to the generation of ATP under conditions of low oxygen concentration. , …”
In this paper we characterize the mechanistic roles of the crystalline purple membrane (PM) lattice, the earliest bacteriorhodopsin (BR) photocycle intermediates, and divalent cations in the conversion of PM to laser-induced blue membrane (LIBM; lambda(max)= 605 nm) upon irradiation with intense 532 nm pulses by contrasting the photoconversion of PM with that of monomeric BR solubilized in reduced Triton X-100 detergent. Monomeric BR forms a previously unreported colorless monomer photoproduct which lacks a chromophore band in the visible region but manifests a new band centered near 360 nm similar to the 360 nm band in LIBM. The 360 nm band in both LIBM and colorless monomer originates from a Schiff base-reduced retinyl chromophore which remains covalently linked to bacterioopsin. Both the PM-->LIBM and monomer-->colorless monomer photoconversions are mediated by similar biphotonic mechanisms, indicating that the photochemistry is localized within single BR monomers and is not influenced by BR-BR interactions. The excessively large two-photon absorptivities (> or =10(6) cm(4) s molecule(-1) photon(-1)) of these photoconversions, the temporal and spectral characteristics of pulses which generate LIBM in high yield, and an action spectrum for the PM-->LIBM photoconversion all indicate that the PM-->LIBM and Mon-->CMon photoconversions are both mediated by a sequential biphotonic mechanism in which is the intermediate which absorbs the second photon. The purple-->blue color change results from subsequent conformational perturbations of the PM lattice which induce the removal of Ca(2+) and Mg(2+) ions from the PM surface.
“…Indeed, even if the transition has been defined at each boundary of the solubilization and/or the reconstitution stages by the molecular composition of the mixed detergent-lipid aggregates and by the detergent concentration in the aqueous continuum, the intermediate structures and the evolution of their compositions throughout the vesicle-to-micelle transition are not yet completely understood. Information about the supramolecular level of the transition has already been obtained by light-scattering measurements, i.e., the determination of the phase boundaries of the vesicle-to-micelle transition (Ollivon et al, 1988;Paternostre et al, 1988;Urbaneja et al, 1990;Inoue et al, 1992;Seras et al, 1992Seras et al, , 1993 and of the solubilization of native biomembranes (del Rio et al, 1991;Meyer et al, 1992;Kragh-Hansen et al, 1993). The structure, the shape, and the size evolution of the mixed aggregates appearing during the transition have been determined by cryotransmission electron microscopy (Edwards et al, 1989;Vinson et al, 1989;Walter et al, 1991).…”
The mechanism of the solubilization of egg phosphatidylcholine containing 10% (M/M) of egg phosphatidic acid unilamellar vesicles by the nonionic detergent, octyl beta-D-glucopyranoside, has been investigated at both molecular and supramolecular levels by using fluorescence and turbidity measurements. In the lamellar region of the transition, the solubilization process has been shown to be first a function of the initial size before reaching an equilibrium aggregation state at the end of this region (the onset of the micellization process). The analysis during the solubilization process of the evolution of both the fluorescence energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine (Rho-PE) and the fluorescence of 6-dodecanoyl-2-dimethylaminoaphtalene (Laurdan) has allowed us to determine the evolution of the detergent partitioning between the aqueous and the lipidic phases, i.e., the evolution of the molar fraction of OG in the aggregates (XOG/Lip) with its monomeric detergent concentration in equilibrium ([OG]H2O), throughout the vesicle-to-micelle transition without isolating the aqueous medium from the aggregates. The curve described by XOG/Lip versus [OG]H2O shows that the partition coefficient of OG is changing throughout the solubilization process. From this curve, which tends to a value of 1/(critical micellar concentration), five different domains have been delimited: two in the lamellar part of the transition (for 0 < [OG]H2O < 15.6 mM), one in the micellization part, and finally two in the pure micellar region (for 16.5 < [OG]H2O < 21 mM). The first domain in the lamellar part of the transition is characterized by a continuous variation of the partition coefficient. In the second domain, a linear relation relates XOG/Lip and [OG]H2O, indicating the existence of a biphasic domain for which the detergent presents a constant partition coefficient of 18.2 M-1. From the onset to the end of the solubilization process (domain 3), the evolution of (XOG/Lip) with [OG]H2O can be fitted by a model corresponding to the coexistence of detergent-saturated lamellar phase with lipid-saturated mixed micelles, both in equilibrium with an aqueous phase, i.e., a three-phase domain. The micellar region is characterized first by a small two-phase domain (domain 4) with a constant partition coefficient of 21 M-1, followed by a one-phase mixed-micellar domain for which XOG/Lip no longer linearly depends on [OG]H2O. The results are discussed in terms of a phase diagram.
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