title: Bio-layer interferometry for measuring kinetics of protein-protein interactions and allosteric ligand effects.
abstract: We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit ε with the catalytic complex of Escherichia coli ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit ε could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of ε undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of ε's conformations is predominant. The assay measures kinetics of ε's binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound ε to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on ε's conformational changes.
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University.
authors.forename: Naman B
authors.initials: NB
authors.lastname: Shah
authors.affiliation: Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University; duncant@upstate.edu.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Duncan
mesh_terms: D001710:Biotin / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D007368:Interferometry / Q000295:instrumentation / Q000379:methods*; D007700:Kinetics; D008024:Ligands; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry
chemical_list: D055767:Immobilized Proteins; D008024:Ligands; D011506:Proteins; D001710:Biotin; D019809:Streptavidin