The earliest events in protein folding are the most crucial for elimination of undesirable final conformations and formation of the native (folded) protein. This work seeks to address a deficit of information on the primary events involved in protein folding as well as address specific questions regarding the overall (global) mechanism of protein folding. The effects of free energy, point mutations, and secondary structure on the folding pathways of horse and yeast iso-1-cytochrome c will be examined. In proteins that contain a redox-active site, such as cyt c, conformation stability generally depends on the oxidation state. The midpoint of equilibrium unfolding curves for the oxidized and reduced forms of protein as a function of denaturant are displaced from one another. There exists a set of conditions in which the protein is folded in one oxidation state and unfolded in the other. Under these conditions, a rapid electron transfer (ET) can be used to trigger either folding or unfolding by generating the more or less stable state. Since ET is one of the fastest known reactions, folding can be triggered at 10-3 to 10-6 seconds or faster. Excitation with a short (i.e. 20 ns) laser pulse will produce a highly reducing photochemical excited state which can then react with the oxidized protein and induce folding. An advantage of this method is that the back reaction, ET from the ferrous protein to the oxidized sensitizer, is necessarily slow due to the low concentrations of these reactive species. This results in a long time window for the observation of folding kinetics. Transient absorption and florescence spectroscopy are employed to follow the dynamics of cyt c folding from the microsecond to second timescale. An important question in the folding pathways of heme-containing proteins centers on the heme iron ligation. In the native, folded protein, the heme iron is ligated by a histidine and methionine. In the unfolded protein the methionine is dissociated from the heme center and there is evidence that it is replaced by histidine. It is not known whether this sixth position drives the folding reaction of follows its. We will assess the importance of non-native heme iron ligation in the folding reaction of cytochrome c. Another important question regarding protein folding is the dependence of the folding rate on the folding free energy. This question can be addressed by examination of the folding kinetics of mutant yeast iso-1-cytochrome c. Mutations of single amino acids rarely lead to substantially different properties of the protein. However, an exception of this general rule is found in residue 52 mutants of yeast iso-1-cytochrome c. In this case, deltaGof is extremely sensitive to the amino acid at that position. We will examine the energetics and folding rate constants of Asn52XXX mutants to determine whether these single point mutations affect the folding rate constants as dramatically as the folding free energies. These mutants give us almost identical proteins with dramatic folding free energy differences; thus, they are ideally suited to examine the dependence of the folding rate constant on the folding free energy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Postdoctoral Individual National Research Service Award (F32)
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Molecular and Cellular Biophysics Study Section (BBCA)
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California Institute of Technology
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United States
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