The enzyme rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) offers and unusual opportunity to study and understand functionally important features of protein structure and its fluctuations. The proposed research is designed to exploit advances made during the previous grant period by using rhodanese as a model for the role of domain interactions and protein flexibility in catalysis and ligand interactions. The overall aims are to continue to pursue correlations between flexible conformational states of rhodanese and the catalytic events in which it participates. The suggested approach is to use substrates and inhibitors to produce stable species which approximate catalytic intermediates, and to study their conformations with chemical, physical and functional methods that are sensitive to structural dynamics. Fluorometry is a central technique because it represents a minimally interactive probe sensitive to subtle and dynamic changes. However it is vital that information from a variety of approaches be correlated with functional studies. A major goal will be to characterize the dynamic properties of rhodanese species with steady-state and nanosecond fluorescence methods using the intrinsic fluorophores as well as a variety of probes. In addition, corroborative techniques that can sense structural dynamics will be used, e.g., tritium exchange, rapid reaction kinetic methods and differential chemical modification. These results will be compared with these from methods that sense average structure, e.g., CD and ultracentrifugation. This information is then to be correlated with measured kinetic behavior and interpreted with the aid of a high resolution x-ray structure that is available. These approaches will be extended to understand the effects of low concentrations of denaturants, pH and ionic strength, all of which have particularly marked effects on both the structure and function of rhodanese. An underlying objective is to develop a set of techniques for the recognition and understanding of protein flexibility and domain interactions in a variety of other functional systems.
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