ABSTRACT
NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.
Subject(s)
Escherichia coli Proteins/physiology , Escherichia coli/metabolism , Membrane Transport Proteins/physiology , Nucleoside Transport Proteins/physiology , Adenosine/analysis , Adenosine/metabolism , Amino Acid Sequence , Animals , Biological Transport/physiology , Circular Dichroism , Escherichia coli/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/isolation & purification , Membrane Transport Proteins/genetics , Membrane Transport Proteins/isolation & purification , Molecular Sequence Data , Nucleoside Transport Proteins/genetics , Nucleoside Transport Proteins/isolation & purification , Oocytes/chemistry , Phylogeny , Protein Structure, Secondary , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sequence Alignment , Spectroscopy, Fourier Transform Infrared , Substrate Specificity , Uridine/analysis , Uridine/metabolism , XenopusABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Glucuronidase/chemistry , Glucuronides/chemistry , Membrane Transport Proteins/chemistry , Nuclear Magnetic Resonance, Biomolecular/methods , Uridine/chemistry , Bacterial Proteins/metabolism , Carbon Isotopes , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Glucuronidase/metabolism , Glucuronides/metabolism , Kinetics , Membrane Transport Proteins/metabolism , Protein Binding , Uridine/metabolismABSTRACT
Obtrusive 13C-backgrounds can be a problem in 13C NMR-based studies of ligand binding to bacterial membrane transport proteins in their natural state in inner membranes. This is largely solved for the bacterial galactose-H+ symport protein GalP by growing the producing organism Escherichia coli on 13C-depleted glucose (13C = 0.07%) as the main carbon source. 13C solid-state NMR-based binding studies for the inhibitor forskolin 1 and the transported substrate glucose 2, both singly labeled with 13C, are reported and discussed. For 1, tight binding is observed, while for 2, significant exchange takes place during the time scale of the NMR experiment.