SOLUTION SAXS STUDIES OF CONFORMATIONAL CHANGES IN MEMBRANE FUSION AAA+ ATPASES

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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. NSF and VCP are two hexameric ATPases that play an essential role in intracellular membrane trafficking. Fusion of lipid bilayers is mediated by proteins present on opposing membranes, known as SNAREs, that associate to form a highly stable complex. NSF uses the energy of ATP hydrolysis to disassemble SNARE complexes so that the components can be recycled for subsequent rounds of fusion. We propose to carry out solution SAXS on NSF and VCP to study conformational changes upon ATP binding, hydrolysis and release. The aims are to 1) generate low resolution models of NSF in its ATP-bound, ADP-bound, and nucleotide-free states using the x-ray structures of the individual domains, and 2) measure changes in scattering as a function of time to obtain kinetic information. By combining the information derived from SAXS data with other biophysical and crystallographic studies underway in our laboratory, we hope to gain an understanding as to how nucleotide hydrolysis and exchange is coupled to the structural changes in NSF and disassembly of SNARE complexes.