This proposal requests biophysical instrumentation and computing equipment to enable a broad interdisciplinary investigation of fundamental aspects of protein folding and protein-ligand interactions. The participating research groups are from the Biomolecular Structure Center and the departments of Biochemistry, Biological Structure, and Bioengineering at the University of Washington. The dramatic increase in the power of computers over the last five years has ushered in a truly exciting new era in structural molecular biology. There is now hope of understanding the multitude of complex interatomic interactions in proteins at the level of protein folding and proteinligand interactions. The development of adequate computational models, however, requires detailed biophysical characterization of relevant experimental systems. We propose to take a combined biophysical and computational approach to understanding the interactions which underlie the structure and function of proteins. Spectroscopic methods will be used to probe the kinetics and thermodynamics of protein folding and protein-ligand interactions in detail. These results will be used to develop computational models that have as their long range goals the understanding of the rules linking amino acid sequences, protein 3-dimensional structures, and protein-ligand interactions. The work described in this proposal has three main thrusts. The first part is focused on understanding the dependence of protein folding on amino acid sequence, using extremely simple model proteins. The second part is focused on the detailed understanding of a model proteinligand interaction: the binding of biotin to streptavidin. The third part seeks to extend knowledge gained in parts one and two to develop more general methods for experimentally and computationally charactelizing a wide valiety of protein ligand interactions, and for understanding the "restricted folding" of a short peptide which is enclosed by a larger multimeri c protein assembly. The links between three areas are not merely conceptual; the biophysical and computational methods required to address all three issues are highly overlapping. In each case the range of scientific questions currently accessible is limited by our current inability to experimentally determine such key kinetic parameters as on-rates and off-rates and to monitor protein folding reactions. We are similarly limited by current computational resources as to the size and complexity of model systems which may be simulated. The requested instrumentation therefore consists of (1) absorption and circular dichroism spectrometers with associated stopped-flow apparatus to follow the thermodynamics and kinetics of protein folding in solution, (2) surface plasmon resonance (SPR) apparatus which allows exquisitely sensitive measurement of the kinetics of protein-ligand binding, and (3) computer equipment to support the theoretical and predictive side of the investigation as well as visualization of the model systems under study.
