Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

Hao Xie, Simon G. Patching, Maurice P. Gallagher, Gary Litherland, Adrian R. Brough, Henrietta Venter, Sylvia Y.M. Yao, Amy M.L. Ng, James D. Young, Richard B. Herbert, Peter J.F. Henderson, Stephen A. Baldwin

Research output: Contribution to journalArticle

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 Km values of ∼20–30 μM 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′-13C]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 α-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.
Original languageEnglish
Pages (from-to)323-336
Number of pages14
JournalMolecular Membrane Biology
Volume21
Issue number5
DOIs
Publication statusPublished - 2004
Externally publishedYes

Fingerprint

Nucleoside Transport Proteins
Pyrimidine Nucleosides
Escherichia coli
Uridine
Oocytes
Purine Nucleosides
Proton-Motive Force
Membrane Transport Proteins
Xenopus laevis
Fourier Analysis
Circular Dichroism
Eukaryota
Affinity Chromatography
Membrane Potentials
Adenosine
Spectrum Analysis
Proteins
Magnetic Resonance Spectroscopy
Metals
Binding Sites

Keywords

  • bacteria
  • solid-state NMR
  • major facilitator superfamily
  • transport
  • Nucleoside

Cite this

Xie, Hao ; Patching, Simon G. ; Gallagher, Maurice P. ; Litherland, Gary ; Brough, Adrian R. ; Venter, Henrietta ; Yao, Sylvia Y.M. ; Ng, Amy M.L. ; Young, James D. ; Herbert, Richard B. ; Henderson, Peter J.F. ; Baldwin, Stephen A. / Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family. In: Molecular Membrane Biology. 2004 ; Vol. 21, No. 5. pp. 323-336.
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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 Km values of ∼20–30 μM 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′-13C]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 α-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.",
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Xie, H, Patching, SG, Gallagher, MP, Litherland, G, Brough, AR, Venter, H, Yao, SYM, Ng, AML, Young, JD, Herbert, RB, Henderson, PJF & Baldwin, SA 2004, 'Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family', Molecular Membrane Biology, vol. 21, no. 5, pp. 323-336. https://doi.org/10.1080/09687860400003941

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family. / Xie, Hao; Patching, Simon G.; Gallagher, Maurice P.; Litherland, Gary; Brough, Adrian R.; Venter, Henrietta; Yao, Sylvia Y.M.; Ng, Amy M.L.; Young, James D.; Herbert, Richard B.; Henderson, Peter J.F.; Baldwin, Stephen A.

In: Molecular Membrane Biology, Vol. 21, No. 5, 2004, p. 323-336.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

AU - Xie, Hao

AU - Patching, Simon G.

AU - Gallagher, Maurice P.

AU - Litherland, Gary

AU - Brough, Adrian R.

AU - Venter, Henrietta

AU - Yao, Sylvia Y.M.

AU - Ng, Amy M.L.

AU - Young, James D.

AU - Herbert, Richard B.

AU - Henderson, Peter J.F.

AU - Baldwin, Stephen A.

PY - 2004

Y1 - 2004

N2 - 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 Km values of ∼20–30 μM 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′-13C]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 α-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.

AB - 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 Km values of ∼20–30 μM 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′-13C]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 α-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.

KW - bacteria

KW - solid-state NMR

KW - major facilitator superfamily

KW - transport

KW - Nucleoside

U2 - 10.1080/09687860400003941

DO - 10.1080/09687860400003941

M3 - Article

VL - 21

SP - 323

EP - 336

JO - Molecular Membrane Biology

JF - Molecular Membrane Biology

SN - 0968-7688

IS - 5

ER -