9727439 Callender The central purpose of this work is to understand the dynamics of protein folding from an experimental point of view by measuring what structures form on what time scales along the folding pathway(s). In order to study the fast (sub-msec) processes that are known to be key to understanding the pathway, novel laser induced temperature jump relaxation spectroscopy with a resolution of about 10 nsec (and down to 50 psec if necessary) are developed and employed. Here the temperature of the water solvent is suddenly raised about 20 (C, and the system responds by relaxing to the new equilibrium condition. IR absorption in the structure sensitive protein amide-I band and fluorescence of tryptophan and extrinsic chromophores are used to probe time dependent changes in structure. Static equilibrium studies using these probes, and others, are also performed. The study will concentrate on the folding of apomyoglobin (apoMb, myoglobin without its heme group) because the protein is relatively simple and serves as an archetype for folding of proteins which are small, single-domain, and globular. Moreover, various well characterized unfolded states of the protein can be prepared and the folding kinetics from one state to another probed. In this way, the kinetics of the folding pathway can be 'peeled' away. In addition, the kinetics of model helical peptides, particularly the Fs peptide, are also to be studied as model systems. The peptides will be isotopically labeled at specific locations so as to unravel position dependent dynamics. Understanding how proteins fold up into their compact three dimensional forms is a crucial problem in modern structural biology. This is so because the particular structure of a protein governs its specific function, and any biological activity is supported by a corresponding protein system. It has been known for quite some time that the spatial structure of a protein molecule is determined completely by the sequence of amino acids of its polypep tide chain, at least for small proteins and probably for all proteins in a general sense. Moreover, the amino acid sequence also codes the way how the three-dimensional structure is reached efficiently. Both aspects are equally important for protein engineering purposes, because any polypeptide sequence coding a new protein should not only offer some new function but also ensure its efficient folding. Otherwise, the expressed protein cannot be accumulated in adequate quantities because of cellular processing. For several decades there has been much effort on determining the kinetics of how a protein folds from it extended, unfolded conformation to its final compact form, asking the questions of what types of structures form first and on what time scales. However, only recently have new techniques, partly developed by these studies, permitted study of the all important fast kinetics involved in protein folding. The overall goal of this research is to measure these fast processes so that it is possible to determine the rules that guide the folding pathway for the first time. ***
