title: Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+-ATPase.
abstract: Vacuolar H+-ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V1-ATPase and membrane-integral V0proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V1V0ND). We show that V1V0ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University 750 East Adams Street, Syracuse, New York, 13210.
authors.forename: Stuti
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University 750 East Adams Street, Syracuse, New York, 13210.
authors.forename: Stephan
authors.identifier: 0000-0002-2721-2789
authors.initials: S
authors.lastname: Wilkens
mesh_terms: D007368:Interferometry; D008051:Lipid Bilayers / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry*
chemical_list: D008051:Lipid Bilayers; D029701:Saccharomyces cerevisiae Proteins; D025262:Vacuolar Proton-Translocating ATPases
keywords: biolayer interferometry; inhibitor binding; lipid nanodiscs; membrane protein; protein-protein interaction; vacuolar ATPase