Thermodynamic and kinetic studies of protein structure and enzymic mechanisms

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(i) Methods are being developed for the detection of very low concentrations of flourescently labeled proteins in the presence of very high concentrations of unlabeled proteins and polymers. These techniques will be used to investigate non-specific protein/protein and protein/polymer interactions in solution. (ii) Optical studies on the structures of amyloid peptides and proteins are continuing.We are particularly interested in origin of the very intense CD spectrum which has been associated with the cross-beta structure of amyloid fibrils. (iii) Riboswitches are functional RNA elements typically located in the 5'-region of m-RNAs, involved in modulation of gene expression. A type II riboswitch from proteobacteria binds magnesium and S-adenosyl methionine (SAM). Previous work has shown that binding of these ligands produces a large decrease in molecular volume (Chen et al, in press). We predicted that these reactions should be very sensitive to the presence of molecular crowding agents. Binding of Mg++ and SAM to the riboswitch induces marked changes in it circular dichroism spectrum. High concentrations of the crowder trimethylamine oxide (TMAO) produces similar changes. Using this change for analysis, we can show that increasing concentrations of TMAO facilitates binding of SAM to the riboswitch.