Increasing experimental evidence suggests that many proteins undergo persistent and rapid global unfolding/refolding cycles in vivo. The extent to which these cycles modulate function or the role they play in human health remains unclear. However, the transient nature of the native state of some important proteins is well documented, and therefore must be considered in any detailed analysis of their function. The long-term goal of this proposal is to elucidate rapid protein folding mechanisms in order to understand the relationship between sequence and folding kinetics, and subsequently to provide insights into the role of protein folding cycles in biology. The studies will focus on monomeric lambda repressor (lambda 6-85), an 80- residue alpha-helical protein that is a paradigm for the ubiquitous class of helix-turn-helix transcriptional regulators. Previous studies have shown that lambda 6-88 samples its globally denatured state 30 times per second, making it an ideal candidate for studying protein folding cycles. The focus of the proposed investigations will be on the reaction mechanism in vitro, in order to understand how to manipulate the mechanism through changes in sequence. A long-range goal is to develop a systematic approach for perturbing the folding mechanism in vivo, particularly for proteins undergoing functionally relevant folding cycles. In addition, as one of the fastest-folding proteins characterized to date, lambda 6-85 is an ideal model for the early events in the folding of slow-folding proteins whose folding prior to the rate-limiting step is difficult to study. The proposed experiments fall into three categories: 1)Studies of the conformation and energetics of kinetically relevant partially folded substates, including NMR and CD studies of models of denatured lambda 6-85 2) Further development of a quantitative model for the folding mechanism of lambda 6-85, through comparison of experimental measurements of folding rates of a variety of variants with predictions of the model 3) Development of experimental strategies to directly detect the elementary steps in lambda 6-68 folding, based on amide hydrogen exchange and time-resolved fluorescence. The proposed research is designed to answer the simple question: Why does lambda 6-85 fold so fast? An answer to this question will permit manipulation of the folding rate in order to answer the corollary: Why was lambda repressor evolved to fold so fast? The answer will play a key role in developing an understanding of protein folding cycles in biology.
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