Despite an astronomical number of possible conformations, most naturally evolved polypeptides have acquired the ability to spontaneously fold into a unique three-dimensional structure, which gives rise to a protein's biological activity. A detailed structural, energetic and kinetic description of the reversible folding process in vitro for a representative set of proteins is an essential step toward understanding the underlying protein folding in the cell, and its medical implications. Because of the limited time resolution of conventional kinetic methods, little is known about the process of folding on the sub-millisecond time scale. These early structural events are the key to understanding how protein folding is initiated and are the main focus of many current theoretical models. Thus, the primary objective of this project is to directly observe the formation of partially folded early intermediates known to appear for many proteins during the first few milliseconds of refolding. A recently developed and extensively tested capillary mixing device makes it possible to extend the time resolution of continuous-flow fluorescence and quenched-flow hydrogen exchange studies into the microsecond time scale. This approach will be used to study the dynamics of early structural events, such as the initial chain collapse, formation of stable hydrogen bonds and long-range tertiary interactions for a series of well-characterized proteins and some mutants, with engineered fluorescence probes. Among these are mitochondrial and bacterial cytochromes c, ubiquitin and staphylococcal nuclease, which have been characterized extensively in Dr. Roder's laboratory by conventional structural and kinetic methods. The results will provide unique insight into the structural and dynamic properties of early structural intermediates and their kinetic role in protein folding.
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