The long-term objectives are to understand the molecular dynamics and functions of integral membrane proteins. Membrane proteins carry out a wide variety of important biological functions. Molecular understanding of their functions is of importance in health and biology. Currently, first steps are being taken in the characterization of a number of such membrane proteins such as bacteriorhodopsin, visual pigments, beta-adrenergic, muscarinic, acetylcholine and other receptors, ionic channels and ATPases and many other transport proteins. Our specific objectives are to develop methods for determining their secondary and tertiary structures. Experimental approaches proposed are: (1), three- dimensional crystallization of membrane proteins. This is urgently needed and it would be a major goal. Crystallization of bacteriorhodopsin and rhodopsin will be undertaken in collaboration with Prof. G. Petsko (M.I.T.) and Prof. L. Delucas (Univ. of Alabama). (2), Methods (chemical, enzymatic and immunological) will be developed for characterization of the extra-membranous peptide loops and for the lengths of the membrane-embedded helical segments. Studies of the loops will involve selective chemical degradation with the Meerwein reagent (triethyl oxonium tetrafluoroborate), interactions with appropriate anti-peptide antibodies and extensive manipulation by recombinant DNA methods to carry out specific labeling and other investigations. (3), Further topographical studies will involve studies of the interactions between the membrane-embedded segments and with phospholipids (protein-lipid interactions). Specific approaches, exemplified with respect to bacteriorhodopsin are (a) mutagenesis to introduce 'reporter' amino acids, e.g. cysteine in different domains and studies of crystal lattices formed from Hg++ derivatives (with D. M. Engelman) or after spin-labeling (with Wayne Hubbell, U.C.L.A.), (b), reconstitution of the proteins from fragments after introducing photoactivatable groups in the fragments and subsequent photolytic crosslinking, (c), crosslinking using phospholipids carrying photoactivatable groups and (d) membrane-soluble photolabeling reagents.
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