Substrate Affinities for Membrane Transport Proteins Determined by ~(13)C Cross-Polarization Magic-Angle Spinning Nuclear Magnetic Resonance Spectroscopy

摘要

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]-β-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 KD value of 4.17 mM for the labeled ligand and 0.42 mM for an unlabeled ligand, p-nitrophenyl β-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 KD 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.