EPR STUDIES OF MOLECULAR MOTION IN MEMBRANE SYSTEMS

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The goal of this proposal is to describe the motion of lipid and protein components in membranes within biological systems and to relate such motion to the exercise of function. Specifically we aim to: a) characterize lipid-protein interactions in membranes, and protein-protein association of "soluble enzymes" with one another and with membranes, in terms of motional behavior of membrane components; b) study factors which modulate such motion; and c) relate motional characteristics of membrane components to the exercise of function. The study of the motion of protein components will emphasize use of satguration transfer EPR (ST-EPR) which can measure motion in the slow time domain, characteristic of macromolecules and supramolecular complexes. Spin labels will be synthesized and used to label membrane components in selective positions to optimize interpretation. The spectra will be analyzed by computer simulation using newly available algorithms for computation of anisotropic motion spectra. As data become available, the application of theory can be tested, refined and extended. Studies involving the motion of lipids will be compared with NMR studies so that the reliability of spin labels can be analyzed. The program will test and extend the general application of EPR and ST-EPR to the study of motional behavior of membrane components. The main systems to be investigated include: a) D-Beta-Hydroxybutyrate dehydrogenase, a lipid-requiring enzyme; b) The CA++ pump of skeletal muscle sarcoplasmic reticulum; the catalytic pumping cycle has been carefully studied and should serve as a prototype to relate motional characteristics to specific functional detail; c) Transhydrogenase from bovine heart mitochondria, consisting of a single membrane polypeptide subunit capable of controlled gneration of protonmotive force; d) ATP synthetase (F1-Fo) from bovine heart mitochonria; this transducer is capable of interconverting protonmotive force to ATP synthesis or hydrolysis. The reconstitution approach is emphasized. Membranes of defined composition and function can be prepared. In this way, the influence of a variety of conditions on membrane composition, function and motional characteristics can be studied and correlated.