The amino acid sequence of a protein uniquely determines its tertiary structure. Deciphering this relationship, the protein folding problem, has become increasingly important to molecular biologists. DNA sequencing has become routine, but structural experiments remain difficult. Two semi-empirical approaches to the folding problem have evolved: detailed energy calculations and the hierarchic condensation model. This grant focuses on the hierarchic condensation model which presumes that secondary structure is a useful computational intermediate in protein folding. We propose to study known structures. Initial efforts will be directed at deducing the rules which govern alpha-helix and beta-strand formation. Secondary structure prediction will follow an """"""""expert systems"""""""" approach which has been successfully applied to locating turns in a variety of proteins (Cohen et al (1986) Biochemistry 25, 266-275). This algorithm will be combined with procedures for exploring possible tertiary structure through the packing of secondary structure units (e.g. Cohen et al (1979) J. Molec. Biol. 132, 275-288). Methods to sort amongst the alternative structures will be developed. These methods hopefully will evolve from the study of the spatial, electrostatic and catalytic properties of proteins of known structure. Finally, these mathematical models will be applied to a variety of biologically interesting macromolecules with known amino acid sequence but unknown tertiary structure. Collaborative ventures will be established to experimentally test the predicted structures for Interleukin-1 (IL1), Interleukin-2 (IL2), and Parathyroid Hormone (PTH). Fragments of the IL1 sequence which correspond to predicted loop regions will be designed. Dr. Dinarello at Tufts will investigate the immunologic relevance and potential antagonist activity of these fragments. With Drs. Ciardelli and Smith at Dartmouth, the structure of IL2 will be examined through site directed mutagenesis. The theoretical effect of a mutation on the protein's stability and activity will be modelled and tested by circular dichroism spectroscopy and receptor binding assays. With Drs. Kuntz, Stewler and Arnaud at UCSF, the structure of PTH and chemical variants of PTH will be characterized. It is hoped that structural modelling will lead to the design of pharmacologically relevant mutant proteins.
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