The role of retinal in the long-range protein-lipid interactions in bacteriorhodopsin-phosphatidylcholine vesicles (original) (raw)
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European Journal of Biochemistry, 1993
Mismatch between the hydrophobic thicknesses of transmembrane proteins and the supporting lipid bilayer and its consequences on the lateral organization of lipids have been investigated with bacteriorhodopsin and phosphatidylcholine species with a variety of acyl-chain lengths. The purple membrane, from the bacterium Halobacteriuin halobium, was used and reconstituted with dilauroyl-(Lau,GroPCho), dimyristoyl-(Myr,GroPCho), dipalmitoyl-(Pam,GroPCho) and distearoyl-(Ste, GroPCho) glycerophosphocholine. The phase behaviour of the lipids was investigated at different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the 1-acyl-2-[ 8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluorescence depolarization of the 1,6-diphenyl-l,3,5-hexatriene probe. Data obtained with 1-acyl-2-[8-(2-anthroyl)-oct~oyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the phase transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic thicknesses and also on the protein concentration and aggregation state in the lipid bilayer. In the region of high protein concentration (bacteriorhodopsidphosphatidylcholine molar ratios = 1 : 50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length d, = 3.0-3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau,GroPCho (AT = 40 K, mean hydrophobic thickness d = 2.4 nm), and to a lesser extent of Myr,GroPCho (AT = 23 K, d = 2.8 nm), accounting for a strong rigidifying effect of the protein on these short-chain lipids. Bacteriorhodopsin had no influence on the phase properties of Pam,GroPCho (AT = 0 K, d 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste,GroPCho was decreased (AT =-13 K, d = 3.7 nm), indicating a fluidifying effect of the protein on this long-chain lipid. Similar effects on the lipid acyl-chain order were observed in the region of high-protein dilution (bacteriorhodopsidphosphatidylcholine molar ratios < 1 : 500). In this region and for Lau,GroPCho, both the spectroscopic data and circular-dichroism spectra indicated that the protein was in the monomeric form. Phase diagrams, in temperature versus bacteriorhodopsin concentration, were constructed for Lau,GroPCho and Ste,GroPCho. On account of microscopic theoretical models and of the relative values of d, and d, these diagrams indicate a preference of the protein for those lipid molecules which are in the gel-ordered state in Lau2. GroPCho but in the liquid disordered state in Ste,GroPCho. The phase diagram of Lau,GroPCho was also analyzed using another theoretical approach based upon elastic models within the Landaude Gennes theory. This allowed for the estimation of the coherence length < which characterizes the distance over which the hydrophobic thickness of the lipid bilayer is perturbed by the protein. A value of 1.2 nm was found, agreeing relatively well with theoretical predictions. Biological membranes contain a large variety of proteins and lipids and despite extensive studies on the structure of these complex assemblies during the last decade, we still have no direct information on the potential specificity of lipid
Biochemistry, 1985
The effect of monomeric bacteriorhodopsin on the lipid order and dynamics in dimyristoylphosphatidylcholine (DMPC) vesicles was monitored as a function of the protein to lipid ratio by timedependent fluorescence anisotropy measurements with diphenylhexatriene (DPH). Energy transfer from the donor DPH to the acceptor retinal of bacteriorhodopsin was used as a spectroscopic ruler to estimate the range of the protein-induced perturbation of the lipid phase. The Forster distance for this donor-acceptor pair is approximately 45 A. Since the effective radius of bacteriorhodopsin is about 17 A, the labels within a neighborhood of radius R , around bacteriorhodopsin are strongly quenched and make a negligible contribution to the end value of the fluorescence anisotropy, from which the order parameter is calculated. Instead, the order parameter is mainly determined by the labels which are more than the Forster distance away from the retinal and which are consequently in the bulk lipid phase. The observed linear increase in order parameter from 0.29 for pure DMPC to 0.62 for a molar bacteriorhodopsin to DMPC ratio of 1/52 thus indicates that the order of the bulk lipids is increased by the interaction with bacteriorhodopsin and that the range of this perturbation is larger than 45 A. In the absence of the acceptor retinal, no energy transfer occurs, and both bulk and boundary lipids are weighted equally in the determination of the order parameter. Only a very small change in the order parameter is observed upon removal of the acceptor, suggesting that bacteriorhodopsin affects the order of all the lipids in roughly the same way. The rotational diffusion constant of DPH determined from the initial slope of the anisotropy decay is independent of the surface concentration of bacteriorhodopsin and of the presence of the acceptor retinal. The viscosity calculated from the rotational diffusion constant is approximately 0.1 P at 35 O C and is an order of magnitude smaller than that determined previously froii the rotational diffusion of bacteriorhodopsin. A comparison of the viscosities determined from the steac pstate and time-resolved fluorescence anisotropy of DPH shows that the first method overestimates the viscosity by as much as a factor of 10.
Biochemistry, 1981
The thermotropic lipid phase transition of dimyristoylphosphatidylcholine vesicles reconstituted with bacteriorhodopsin was investigated as a function of the lipid to protein ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6diphenyl-l,3,5-hexatriene. Two attractive features of this system are that the lipid phase transition induces lipid-protein segregation and that the state of aggregation of the protein is known. Above the lipid phase transition and above molar lipid to protein ratios of about 100, bacteriorhodopsin is monomeric. Well below the phase transition, bacteriorhodopsin is aggregated in a hexagonal protein lattice. With increasing amounts of incorporated bacteriorhodopsin, the calorimetric transition broadens, and a second component develops at a temperature which is lower than that of the unperturbed transition. The latter transition was assigned to the disag-
Journal of Molecular Biology, 1978
Bacteriorhodopsin has been incorporated into large unilamellar lipid vesicles. Its aggregation behaviour was investigated using X-ray diffraction, electron microscopy, circular diehroism and rotational diffusion measurements. At temperatures below the lipid phase transition, bacteriorhodopsin crystallizes into patches ~dth the same hexagonal lattice observed in the purple membrane. Above the phase transition, the lattice disaggregates and the protein molecules are monomeric provided the lipid to protein ratio is sufficiently high. 284 R. J. CHERRY ET AL.
Lateral segregation of proteins induced by cholesterol in bacteriorhodopsin-phospholipid vesicles
Biochimica et Biophysica …, 1980
Bacteriorhodopsin in dimyristoylphosphatidylcholine vesicles is randomly distributed in the plane of the membrane and exhibits rotational diffusion above the gel to liquid-crystalline phase transition. Incorporation of cholesterol results in loss of rotational mobility of bacteriorhodopsin, which on the basis of electron microscopy and CD measurements can be assigned to the formation of protein aggregates. It is concluded that bacteriorhodopsin is soluble in the fluid phosphatidylcholine phase but segregates when cholesterol is present in the lipid bilayer.
Biophysical Journal, 1997
A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.
Biophysical Chemistry, 1995
From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC,,,o), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC,,:,, diCIko and diC,& and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidylglycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bilayer thickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array.
Retinal Binding during Folding and Assembly of the Membrane Protein Bacteriorhodopsin †
Biochemistry, 1996
The factors driving folding and assembly of integral membrane proteins are largely unknown. In order to determine the role that the retinal chromophore plays in assembly of bacteriorhodopsin, we have determined the kinetics and thermodynamics of retinal binding during regeneration of bacteriorhodopsin, from denatured apoprotein, in Vitro. Regeneration is initiated by rapid, stopped-flow, mixing of the denatured apoprotein bacterioopsin in sodium dodecyl sulfate micelles with mixed detergent/lipid micelles containing retinal. Regeneration kinetics are measured by time-resolving changes in protein fluorescence. The dependence of each kinetic component on retinal concentration is determined. Only one experimentally observed rate constant is dependent on retinal concentration, leading to identification of only one second-order reaction involving retinal and bacterioopsin. This reaction occurs after a ratelimiting step in bacterioopsin folding, and results in formation of a noncovalent retinal/protein complex. The free energy change of this retinal binding step is determined, showing that thermodynamic information can be obtained on transient intermediates involved in membrane protein regeneration.
Biochemistry, 1979
In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.