The comprehensive goal of this proposal is to determine the mechanisms by which proteins fold into their native three dimensional structures. Many fully unfolded proteins spontaneously refold when placed in the proper environment indicating that the information needed to form the three- dimensional native structure is contained in their amino acid sequences. However, the rules that determine how sequence directs the folding are unknown. To further address the protein folding problem it is necessary to determine the structures and kinetics of the intermediates in the folding pathways. Previous work has indicated that """"""""misfolded"""""""" states can significantly influence a protein's progress toward formation of the native structure. When prevented from misfolding formation of the native structure can become very fast, on the order of 1-20 ms. Further, many proteins are known to exhibit """"""""burst phases"""""""" of refolding that occur in less than a few milliseconds. Consequently it would be of value if techniques were developed that could follow ultrafast events in protein folding and could be capable of providing structural information on these early intermediates. Typical mixing methods (eg stopped flow) currently in use are too slow to allow these measurements and are limited by instrument response times. Although selected faster measurements have been carried out there are few of these studies published and the applicability of these methods is very limited. What is proposed here is a more generally applicable expansion of modern optical methods to include submillisecond kinetics measurements of protein folding. The measurements make use of rapid mixing technology developed in the investigator's laboratory to allow for spectroscopic studies as early as 100 m sec after initiation of the folding process. The specific measurements to be done include: resonance Raman spectroscopy to follow changes in the axial coordination of the heme; UV Resonance Raman to utilize tyrosine and tryptophan residues as reporters of the local environment about their side chains; fluorescence lifetime studies of tryptophan side chains to gain a measure of distance from trp to the heme as well as a measure of the size of the protein; UV-circular dichroism to measure the extent of alpha helix formation; and IR and raman spectroscopies to study secondary structure by deconvolution of amide stretching bands.
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