Substrate affinities for membrane transport proteins determined by 13C cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy.
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.
Patching
Simon G
SG
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, Sackville Street, Post Office Box 88, Manchester M60 1QD, United Kingdom.
Brough
Adrian R
AR
Herbert
Richard B
RB
Rajakarier
J Anton
JA
Henderson
Peter J F
PJ
Middleton
David A
DA
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
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