title: Substrate affinities for membrane transport proteins determined by 13C cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy.
abstract: We have devised methods in which cross-polarization magic-angle spinning (CP-MAS) solid-state NMR is exploited to measure rigorous parameters for binding of (13)C-labeled substrates to membrane transport proteins. The methods were applied to two proteins from Escherichia coli: a nucleoside transporter, NupC, and a glucuronide transporter, GusB. A substantial signal for the binding of methyl [1-(13)C]-beta-d-glucuronide to GusB overexpressed in native membranes was achieved with a sample that contained as little as 20 nmol of GusB protein. The data were fitted to yield a K(D) value of 4.17 mM for the labeled ligand and 0.42 mM for an unlabeled ligand, p-nitrophenyl beta-d-glucuronide, which displaced the labeled compound. CP-MAS was also used to measure binding of [1'-(13)C]uridine to overexpressed NupC. The spectrum of NupC-enriched membranes containing [1'-(13)C]uridine exhibited a large peak from substrate bound to undefined sites other than the transport site, which obscured the signal from substrate bound to NupC. In a novel application of a cross-polarization/polarization-inversion (CPPI) NMR experiment, the signal from undefined binding was eliminated by use of appropriate inversion pulse lengths. By use of CPPI in a titration experiment, a K(D) value of 2.6 mM was determined for uridine bound to NupC. These approaches are broadly applicable to quantifying binding of substrates, inhibitors, drugs, and antibiotics to numerous membrane proteins.
authors.affiliation: Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Sackville Street, Post Office Box 88, Manchester M60 1QD, United Kingdom.
authors.forename: Simon G
authors.initials: SG
authors.lastname: Patching
authors.forename: Adrian R
authors.initials: AR
authors.lastname: Brough
authors.forename: Richard B
authors.initials: RB
authors.lastname: Herbert
authors.forename: J Anton
authors.initials: JA
authors.lastname: Rajakarier
authors.forename: Peter J F
authors.initials: PJ
authors.lastname: Henderson
authors.forename: David A
authors.initials: DA
authors.lastname: Middleton
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005966:Glucuronidase / Q000737:chemistry* / Q000378:metabolism; D020719:Glucuronides / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D014529:Uridine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D020719:Glucuronides; D026901:Membrane Transport Proteins; C088081:NupC protein, Bacteria; C490231:NupC protein, E coli; D005966:Glucuronidase; D014529:Uridine