The mechanism by which a protein folds, given only the information in the primary sequence, remains one of the most difficult and challenging problems in biochemistry.
The specific aims of this proposal are to approach this problem by determining the nature and structural properties of intermediates on the folding pathway. Several methods are to be used. In one, we will use stopped flow NMR techniques to measure the properties of intermediates in real time during the folding process. Site directed mutagenesis coupled with incorporation of fluorine labeled amino acids and a stopped flow NMR device we have built will allow examination of specific regions of a protein during folding. Initially we will study the E. coli dihydrofolate reductase and the intestinal fatty acid binding protein by this method. The NMR studies will be compared with hydrogen/deuterium exchange studies being conducted on both these proteins. Dihydrofolate reductase is an example of alpha/beta structure while the fatty acid binding protein is almost totally a beta sheet protein. In a second approach we will modify or replace structural elements (turns, helices or strands) in the fatty acid binding protein to explore the role of these elements in the folding process. To explore further the nature of intermediates in folding, the interaction between the eukaryotic dihydrofolate reductase and the bacterial chaperonin GroEL will be examined by similar techniques. Since it catalyzes an initial step in nucleotide synthesis, dihydrofolate reductase is the target for numerous chemotherapeutic, antibacterial and antiparasitic drugs. The intestinal fatty acid binding protein is one of a family of proteins that differ in ligand specificity (fatty acids, retinoids and bile salts) and tissue specificity and are found to be useful models for spatial and temporal differentiation in the gut.
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