Fundamental Processes in the Folding of Helical Proteins

Suny At Stony Brook, Stony Brook NY

Investigators

Abstract

This project will provide insight into fundamental aspects of the protein folding problem and will generate important benchmarks for theoretical and computational studies. The potential synergy between theory, simulation and experiment has spanned a tremendous interest in small helical proteins that fold very rapidly. They are excellent model systems for addressing fundamental issues in the folding process, in part because they are tractable targets for all atom simulations and in part because they are also good models for the first steps in the assembly of more complex structures. Experimental studies of several helical proteins will be undertaken. The folding of the Villin headpiece helical subdomain (HP-36) has been the subject of numerous computational and theoretical studies but virtually nothing is known experimentally about its folding mechanism. Site directed mutagenesis will be combined with the incorporation of specific isotopic labels and rapid kinetic techniques to develop a detailed picture of the folding of HP-36. The role of unfolded state structure in rapid folding is a controversial topic. The unfolded state of HP-36 contains significant structure thus it is an excellent model system for addressing this topic. Structure in the unfolded state of HP-36 will be characterized using NMR. The kinetic consequences of mutations that alter the denatured state structure of HP-36 will be examined. An additional goal of the project is to test how rapid folding is altered by context. Arguments have been made that rapid folding and unfolding could be biologically significant but all fast folding proteins studied to date are either small designed proteins or fragments of larger systems. None have been studied in the more biologically relevant context of their intact parent structures. HP-36 is the C-terminal subdomain of the larger Villin headpiece (HP-67). The folding of HP-36 in isolation and when it is part of HP-67 will be compared in order to determine if its rapid folding and unfolding is affected by context. The final objective of the project concerns downhill folding, i.e. folding in the absence of a free energy barrier. Experiments will be conducted to test one of the more striking theoretical predictions of the new view of folding, namely that stabilizing very fast folding proteins can lead to downhill folding. Rationally designed hyperstable variants of HP-36 will be used in these studies. The project will provide broad interdisciplinary training in protein biophysics and protein chemistry to both graduate and undergraduate students. Undergraduate participation and undergraduate curriculum development are important parts of the project. The first steps have already been taken; the content of the large first year undergraduate general chemistry course has been revised to include material on modern biological chemistry. New initiatives will include the development of a one semester advance undergraduate course in chemical biology and biophysical chemistry.