Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching

The translational diffusion of bovine rhodopsin, the Caz+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein 1 3000/1) in liquidcrystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X cm2/s. In consideration of the sizes of the membranebound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbriick, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3 1 1 1-3 1 131. Lipid diffusion was also examined in the same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Trauble, H., & Sackmann, E. (1972) J. Am. Chem. SOC9. 4,4499-45101 may have to be invoked to explain the lipid diffusion behavior

Kinetics of the incorporation of cytochrome b5, an integral membrane protein, into unilamellar dimyristoyllecithin liposomes

The kinetics of incorporation of proteins into phospholipid liposomes were studied using fluorescence of the protein tryptophan residue; fluorescence was detd. at varying protein/lipid ratio and at varying temp. Cytochrome b5, purified from liver microsomes, was exposed to unilamellar dimyristoyllecithin liposomes in a fluorescence cuvette. Tryptophan fluorescence of cytochrome b5 increased rapidly as the protein was incorporated into the lipid bilayer. Protein incorporation was biphasic at low lipid/protein ratios, with an initial fast non-exponential phase followed by a slow phase. The fluorescence was detd. at 15, 25, and 35 Deg, and the rigidity of the liposome bilayer was studied as a function of temp. At low temp (15 Deg), protein incorporation was very slow; however, as the lipid phase transition temp. was approached, protein incorporation increased considerably faster. Thus, there is a pos. correlation between liposome fluidity and the ease of protein incorporation into the bilayer. A model is presented. [on SciFinder (R)