OLIGOMERIC STATE OF VPS4: HIV

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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. The Vps4 protein is the AAA ATPase that drives progression through the vacuolar protein sorting pathway. This pathway delivers substrates and resident enzymes to lysozome precursors. HIV and other enveloped viruses recruit this pathway to bud virus particles from host cells. We recently published the structure of Vps4 in a monomeric state (EMBO J. 24, 3658-69, 2005). Because the protein functions as an oligomer, we now seek to test models for the assembled state. Leading models include 12-mer, 10-mer, 6-mer, or 5-mer, with either single or double disk configurations. We routinely prepare highly pure samples that are soluble to at least 20 mg/ml. Preparations take just a few days to complete. Under appropriate conditions, such as bound nucleotide analog, the protein elutes from a sizing column as a well defined oligomer. The retention time corresponds to a ~9-11 mer, but of course, retention time is highly dependent upon molecular shape, and we seek better estimates of solution molecular mass and shape.