BIOPHYSICAL CHARACTERIZATION OF LIPID-PROTEIN AND LIPID-LIPID INTERACTIONS IN MODEL AND RECONSTITUTED MEMBRANES USING STATIC AND REAL-TIME X-RAY DIFFRACTION AND FLUORESCENCE QUENCHING TECHNIQUES

(1) The dependence of function and lipid binding affinity of an integral transport protein on the fatty acyl chain characteristics of a membrane-forming phospholipid have been determined. Lipid-protein interactions were examined using a fluorescence quenching technique employing spin-labelled phosphatidylcholine (PC). These studies show that the Ca('2+) ATPase from rabbit sarcoplasmic reticulum binds with equal affinity a variety of PC's used to reconstitute the protein into enzymatically active vesicles, regardless of fatty acyl chain length or details of unsaturation. In parallel with the lipid binding studies, the sensitivity of both catalytic activities of the Ca('2+) ATPase to acyl chain characteristics of the PC membranes in which the enzyme was reconstituted has been measured. The enzyme is sensitive only to the effective acyl chain length, which determines the thickness of the bilayer in which the protein is inserted and displays little sensitivity to such details of unsaturation as degree, position and isomeric type.; (2) To estimate the distribution of a membrane protein between gel and liquid crystal phases in a lipid bilayer using the fluorescence quenching technique, the phase behaviour of the lipids in that membrane must be known. To this end, the phase diagram of hydrated unoriented samples of egg phosphatidic acid (egg PA) with (a) egg PC, (b) dimyristoyl PC and (c) dipalmitoyl PC was determined by the method of x-ray diffraction using synchrotron radiation as the x-ray source. The phase properties of egg PA were unaffected by pH in the range 6-8 or by high concentrations of Na('+), Li('+), K('+), Cs('+), F('-), Cl('-), Br('-) and I('-). Ca('2+), Mg('2+), Sr('2+) and Cd('2+), all induced gel phase formation in the egg PA/egg PC system with Cd('2+) and Sr('2+) effecting the more complete isothermal transformation.; (3) The x-ray diffraction pattern of unoriented hydrated dipalmitoyl PC membranes through the thermotropic gel/liquid crystal phase transition has been observed and recorded in real-time. Synchrotron radiation was used as the x-ray source and the dynamic display of the diffracted image was effected using a 4-stage image intensifier tube coupled to an external fluorescent screen. An upper bound of 2s on the transition has been determined. The general applicability of this newly developed method has been demonstrated in polyethylene and rubber stretching, crystaline polyethylene annealing and clay mineral hydration/dehydration experiments.