Quantitative analysis of lipid-lipid and lipid-protein interactions in membranes by use of pyrene-labeled phosphoinositides.

The lateral and rotational dynamics of phosphoinositides and their interactions with proteins were characterized using pyrene-labeled lipid analogues. In these systems, the collision frequency of pyrene-labeled lipids was studied by monitoring the monomeric pyrene fluorescence yield as a function of their mole fraction in the membranes. From this dependence, the lateral diffusion coefficient and a repulsion factor between two pyrene phosphoinositides could be estimated by applying an extended form of the Milling Crowd model [Eisinger, J., Flores, J., & Petersen, W. P. (1986) Biophys. J. 49, 987-1001]. The repulsion appeared to be highly dependent on the amount of negative charge of the lipid headgroups. From experiments with dioleoylphosphatidylcholine vesicles containing band 3 protein, the fraction of lipid molecules bound to this protein and the minimum number of sites possessing affinity for phosphatidylinositol-4-phosphate could be approximately estimated. The results of this study indicate that phosphoinositides are located preferentially adjacent to band 3. Intramolecular excimer formation of dipyrene-labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylinositol-4-phosphate yielded information about the acyl chain dynamics of lipids surrounding the protein and of lipids in the bulk membrane. Time-resolved measurements of the pyrene fluorescence anisotropy showed that in membranes of resealed erythrocyte ghost cells the rotational freedom of pyrene-labeled phosphatidylinositol-4,5-bisphosphate is smaller than that of pyrene-labeled phosphatidylcholine. In contrast, no significant differences could be detected when these pyrene lipids were dispersed in dioleoylphosphatidylcholine membranes. It is proposed that the nonrandom distribution of the phosphoinositides induced by lipid-lipid repulsion and protein-lipid attraction will have a profound effect on the phospholipase C-catalyzed hydrolysis of the phosphoinositides into second-messenger molecules.