This is a proposal to investigate the structure and characteristics of the transition state in the dimerization/folding reaction of the GCN4-p1 peptide. Most models describing the folding pathways of proteins involve formation of secondary structure as a key component. The observation of kinetic intermediates that have secondary structure has been taken as strong support for these proposals. However, in preliminary experiments presented by Dr. Sosnick, it would appear that the transition state in the GCN4-p1 folding reaction, which is an encounter complex between two peptides leading to the homodimer, is lacking in helical character and has conformational flexibility similar to that of the unfolded peptide. These surprising results need to be clarified and enlarged. It is crucial to understand what, if not secondary structure, does guide the initial folding steps, and what features of the transition state allow it to proceed in an energetically downhill manner to the folded structure. The importance of specific structural/energetic features in early pathway steps will be studied with the simple coiled coil zipper, which provides a simple platform on which substitutions can be made that focus on specific structural features. Residues will be substituted that alter stability of these features, and folding rates will be measured using stopped flow spectroscopy. If a structural feature is fully formed at a substituted site in the transition state, then the free energy of the transition state and the equilibrium native stability will be altered equally. The unfolding rate will not change, and only the folding will be affected. For example, to test for the presence of helix, exterior residues having altered helix propensity will be substituted. If helix is present at the rate limiting step, only folding rates will be affected. The capability of focusing on particular features is very important and potentially represents a major advance in our ability to dissect the role of particular structural features in kinetic folding. This strategy will be applied to the folding of both the dimeric and cross-linked versions of the zipper, to test the effects of proximity, collision frequency and chain topology on folding.
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