This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteins in solution exhibit an ensemble of interconverting structures accessible to one another through low energy pathways. The breadth and form of this ensemble defines the dynamic behavior of the protein. It is increasingly clear that protein structure-function relationships must account for this native ensemble and its associated dynamics. Characterization of the native ensembles of a protein represents a significant technical challenge, and although recent advances in NMR, x-ray crystallography, and computational approaches have contributed substantially, new approaches are badly needed. We have demonstrated that wide-angle x-ray solution scattering (WAXS) can be used to estimate the breadth of the native ensemble of proteins in solution. Recent improvements in protocols for collecting and processing WAXS data have resulted in a substantial improvement in accuracy and reproducibility. These advances provide the opportunity to accurately measure second order effects in the scattering patterns making possible their interpretation in terms of changes in the form and breadth of the ensemble of macromolecular structures in solution. Here we will use WAXS to characterize the native state ensemble of a number of representative proteins and study how changes in ligation state, environment and functionally relevant mutations affect the ensemble. Proteins to be studied include hemoglobin, HIV protease, glutamate receptor and lysozyme. We will show that WAXS can be used to estimate the amplitudes of a protein's normal modes and thereby act as an important new tool for establishing a direct link between a protein's environment or ligation state and the relative motions of specific structural elements within it.
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