Our overall goal is to understand the regulation of protein folding and stability. How the amino acid sequence determines the higher order structure of a protein is the weakest link in our understanding of the causal chain that connects the genetic code with biological function. Folding of a protein is affected by its environment, by other molecules and by post-translational modification. Even in the test tube, many proteins do not refold easily and/or are unstable; this has been proposed to depend on aggregation or misfolding of intermediates. Very little information is available about properties of folding intermediates. Proteins called chaperonins (also called cpn, groESL, heat-shock, or hsp) have been implicated to be involved in folding and compartmentalizing other proteins. The enzyme rhodanese (thiosulfate-sulfurtransferase) is ideal for an in vitro study of these issues because: (i) it is a mitochondrial enzyme, is synthesized in the cytoplasm, and retains its N-terminal leader sequence; (ii) it can be reversibly refolded using detergents, and folding intermediates can be isolated and (iii) chaperonins (E. coli cpn10 and cpn60), homologous to mitochondrial hsp proteins, can substitute for detergents and make folding ATP dependent. We anticipate that we can make progress in the practical control of protein folding and stability in vitro and begin to understand the control of folding in the cell.
The Specific Aims are designed to test three hypotheses: I. Sequential conformational changes accompany reversible unfolding of rhodanese. This will be tested by monitoring conformational changes in different parts of the structure during reversible unfolding using fluorescence, chemical modification, and limited proteolysis. The role of the N-terminal """"""""leader sequence"""""""" will be probed using synthetic peptides and a molecular biological approach.
This aim will provide experimental detail about different stages of the folding process, and permit us to test our model for rhodanese folding. II. Folding intermediates are flexible, and sensitive to oxidation. To test this we will characterize, in detail, intermediates that can be stabilized at different stages of the folding process. Conformational dynamics and structure will be measured as in I, as well as with tritium exchange and site-specific antibodies. Site directed mutagenesis will be used to replace two cysteines that give oxidatively trapped conformers. III. Chaperonins assist folding by binding intermediates and preventing aggregation. Physico-chemical methods will identify: (a) conformations of rhodanese in complex with cpn60; (b) the similarity between cpn60- and detergent-complexes of rhodanese; and (c) which parts of rhodanese are involved in and/or respond to complex formation. We will characterize hydrophobic binding sites that we detected on cpn60. Finally, we will test the idea that if aggregation is controlled, chaperonins will participate in multiple turnovers and act as enzymes.
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