OPTICAL ROTATORY POWER OF BIOPOLYMERS

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The long-range objective is to study the conformation of a few proteins and to understand their structure-function relationship. The specific aims for the next three years are threefold. (1) Further tests of CD analysis of protein conformation. At least three methods are currently used for estimating alpha-helix, beta- sheet and beta-turn in a protein molecule. Several problems should still be addressed: the arbitrary choice of the number and kind of proteins; lack of uniform criteria for identifying secondary structures of proteins from x-ray data; the counting of each conformation segment by the number of peptide bonds (based on CD theory) vs. amino acid residues; the question of introducing the two constraints in CD analysis (1>fi>0 and sigma fi=1, where fi is the fraction of the i-th conformation); and the lower limit of attainable wavelength for CD. (2) Helix folding of short polypeptides. Helices are stabilized by nonpolar, ion-pair and charge-helix dipole interactions. Solvent-exposed helical segments of a set of proteins will be surveyed from their x-ray diffraction data and the frequencies of occurrence of charge-helix dipole and ion-pair interactions will be compared. One or two short polypeptides will be chosen to test the helix dipole model and the ion-pair formation. Their conformation in aqueous solution will be studied by varying temperature, pH and ionic strength of the so- lution. These peptides will be prepared either by limited proteolysis of the parent protein or by standard synthesis. (3) Conformation studies of acetylcholinesterase. The folding of structural domains and the topology of the active center of this enzyme will be our major concern. The active center is composed of an esteratic, an anionic and a hydrophobic site. Limited proteolysis will be applied to this enzyme; whether each subunit has more than one domain and, if so, whether the active center is confined within one domain or, more likely, involves more than one domain will be determined. With this information fragments of the enzyme will be synthesized by recombinant DNA technique and their conformation and possible enzymatic activity will be determined. Site-directed mutagenesis can be used to locate the possible binding site for quaternary ammonium ligands and the essential histidine near the esteratic site. The role of the three intrachain disulfides in the conformation stability of the enzyme will be studied by selectively replacing the half cystines with another amino acid again through mutagenesis. A combination of limited proteolysis and recombinant DNA technique will provide useful information about this synaptic enzyme.