REPEAT-PROTEINS; STABILITY, FOLDING KINETICS & EVOLUTION

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DESCRIPTION (provided by applicant): In all branches of life, there are a variety of proteins that contain short, tandemly repeated sequences. Each repeat-sequence folds up into a closed loop containing helices, beta-strands, or a mix of secondary structure units. Adjacent repeats then pack against each other to create an elongated, linear domain with a regular, modular architecture. These repeat-proteins usually participate in protein-protein interactions, often at the center of important biological processes such as signal transduction, and are often associated with disease states such as cancer and bacterial pathogenesis. In the research outlined here, biophysical studies will be performed on repeat-proteins to better understand their folding and their stability. Our experiments will take advantage of the linear architecture of these proteins to answer questions that are difficult to address with non-repeat proteins. We will determine the distances over which repeats can couple with one another, and measure the end-to-end stability distribution in these proteins. We will use deletion experiments to build an "energy landscape" with single-repeat resolution. We will then examine how this "energy landscape" relates to folding rates by studying the folding kinetics of repeat-protein fragments and by determining which repeats are structured in the rate-limiting steps in folding, and will test models that relate folding rates to various structural and energetic features. Owing to their linear, repeated architecture, repeat-proteins seem likely targets for rapid evolution through insertion and deletion. We will evaluate the outcome of such rearrangements by studying the effects on stability of different types of deletions and duplications. We will test whether the fusion of different types of repeat-proteins can adopt a stable fold. The finding that such rearranged proteins are stable would support the idea that functional diversity can be produced by recombination of genes encoding repeat-proteins. We will also seek support for the hypothesis that repeat-proteins appeared early in protein evolution by computer analysis of protein sequences and structures.