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Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H + ‐ATPase
Author(s) -
Sharma Stuti,
Wilkens Stephan
Publication year - 2017
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3143
Subject(s) - nanodisc , biotinylation , chemistry , biochemistry , cytosol , atpase , membrane protein , integral membrane protein , lipid bilayer , biophysics , membrane , vacuole , enzyme , biology , cytoplasm
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 V 1 ‐ATPase and membrane‐integral V 0 proton 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 ( V 1 V 0 ND). We show that V 1 V 0 ND 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.