UNFOLDED STATES AND FOLDING PATHWAYS BY NMR

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DESCRIPTION (Adapted from applicant's abstract): Studies of the structure and dynamics of unfolded states of proteins have assumed great importance for the insights they provide into the initiation of protein folding. In addition, a number of disease states from defects in the folding and stability of specific proteins and an have recently been shown to arise increasing number of key intracellular proteins appear to be unfolded in their biologically functional states. The major goals of this project are focused on the elucidation of folding pathways and the structural characterization of unfolded states. There are three major specific aims, representing an integrated approach that utilizes recently-developed NMR methods: (1) Heteronuclear NMR methods will be used to obtain a quantitative description of the conformations sampled by two unfolded proteins, acid-unfolded apomyoglobin and low-salt-unfolded apoplastocyanin, which contain almost purely a single type of secondary structure in the folded state. The unfolded states are tractable according to preliminary results, and apoplastocyanin in particular has the added advantage that the unfolded form is present under "folding" conditions, neutral pH and ambient temperature, making the results highly applicable-to the protein folding process in vivo and in vitro. A detailed study of backbone structural propensities and polypeptide dynamics in the unfolded state of these two proteins will be made, as well as of denaturants on the structure in the unfolded state. (2) The folding of apoplastocyamin is slow on the NMR timescale and an MR study of the folding process in real time is proposed. The early, fast folding steps will be followed by multiple-step stopped-flow fluorescence and hydrogen exchange pulse labeling. 3) A long-term goal is to extend the work on the unfolded state to include studies of interactions with chaperones. The interaction of unfolded proteins and protpetides will be studies with a small domain from the E. coil chaperone protein DnaJ, which has been shown to bind zinc and to resemble the DNA-binding domain of glucocorticoid receptors. This surprising result typifies the new information that is constantly being uncovered in the protein folding and assembly a continual source of new and important information.