Early Events in Protein Folding

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9507280 Fink The goal of this research is to measure the rate at which an unfolded protein becomes compact, when placed under native conditions, and by comparison with the rates of secondary and tertiary structure formation, to develop a more complete picture of the folding process. Protein folding is currently believed to be a process in which the unfolded protein initially collapses rapidly to a species with unstable secondary structure, followed by transformation into intermediates with stable secondary structure and ultimately the tightly-packed native state. The specific questions we plan to answer are: How fast does the unfolded protein initially become compact, and how does the compactness change as folding progresses from the initial intermediates to the native state? We will measure the rate at which an unfolded protein becomes compact during the refolding process using two techniques: small-angle x-ray scattering and fluorescence energy transfer; these are among the few techniques available for the measurement of the size of proteins in solution. Recent technical improvements in small-angle X-ray scattering instrumentatimn have now made it feasible to carry out the proposed experiments. The proteins to be investigated initially are cytochrome c, staphylococcal nuclease and DnaK. Clearly, knowledge of the rate at which the protein collapses is very important and will help distinguish competing models for folding, and should significantly increase our understanding of the process of protein folding and thus benefit many fields involving proteins. %%% The question of now proteins fold into their native three-dimensional structures is an important one from may perspectives. This research is aimed at answering one aspect of the overall protein folding problem, namely, how fast does the protein become compact, and how does the rate of collapse compare to the other structural events occurring? We propose to use small-angle X-ray scattering and fluorescence energy transfer to mo nitor the size of a folding protein as a function of time using stopped-flow rapid mixing. Small-angle X-ray scattering provides information about the overall size and shape of the molecule; in particular, we will use mostly the radius of gyration to monitor the size changes. The fluorescence energy transfer will involve donor and acceptor groups in distant regions of the protein sequence, so that in the unfolded sate they are far apart and little energy transfer occurs, whereas as the molecule becomes more compact during folding, they become closer together and the fluorescence energy transfer efficiency increases. The proteins to be investigated initially are cytochrome c, staphylococcal nuclease and DnaK. ***