Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate

Chemistry

Text available via DOI:

https://doi.org/10.1021/ja075584k
Final published version

Keywords

Binding Sites, Biochemistry, Calcium-Binding Proteins, Carbon, Escherichia coli, Forskolin, Glucose, Ligands, Magnetic Resonance Spectroscopy, Mass Spectrometry, Monosaccharide Transport Proteins, Mutation, Periplasmic Binding Proteins, Substrate Specificity, Temperature, Tryptophan

View graph of relations

Research output: Contribution to Journal/Magazine › Journal article › peer-review

Published

Standard

Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate. / Patching, Simon G; Henderson, Peter J F; Herbert, Richard B et al.
In: Journal of the American Chemical Society, Vol. 130, No. 4, 30.01.2008, p. 1236-44.

Research output: Contribution to Journal/Magazine › Journal article › peer-review

Bibtex

@article{c3469ae0a2ce4b5c9d4047ff5beba94a,

title = "Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate",

abstract = "An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.",

keywords = "Binding Sites, Biochemistry, Calcium-Binding Proteins, Carbon, Escherichia coli, Forskolin, Glucose, Ligands, Magnetic Resonance Spectroscopy, Mass Spectrometry, Monosaccharide Transport Proteins, Mutation, Periplasmic Binding Proteins, Substrate Specificity, Temperature, Tryptophan",

author = "Patching, {Simon G} and Henderson, {Peter J F} and Herbert, {Richard B} and Middleton, {David A}",

year = "2008",

month = jan,

day = "30",

doi = "10.1021/ja075584k",

language = "English",

volume = "130",

pages = "1236--44",

journal = "Journal of the American Chemical Society",

issn = "0002-7863",

publisher = "AMER CHEMICAL SOC",

number = "4",

}

RIS

TY - JOUR

T1 - Solid-state NMR spectroscopy detects interactions between tryptophan residues of the E. coli sugar transporter GalP and the alpha-anomer of the D-glucose substrate

AU - Patching, Simon G

AU - Henderson, Peter J F

AU - Herbert, Richard B

AU - Middleton, David A

PY - 2008/1/30

Y1 - 2008/1/30

N2 - An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.

AB - An experimental approach is described in which high resolution 13C solid-state NMR (SSNMR) spectroscopy has been used to detect interactions between specific residues of membrane-embedded transport proteins and weakly binding noncovalent ligands. This procedure has provided insight into the binding site for the substrate D-glucose in the Escherichia coli sugar transport protein GalP. Cross-polarization magic-angle spinning (CP-MAS) SSNMR spectra of GalP in its natural membrane at 4 degrees C indicated that the alpha- and beta-anomers of D-[1-(13)C]glucose were bound by GalP with equal affinity and underwent fast exchange between the free and bound environments. Further experiments confirmed that by lowering the measurement temperature to -10 degrees C, peaks could be detected selectively from the substrate when restrained within the binding site. Dipolar-assisted rotational resonance (DARR) SSNMR experiments at -10 degrees C showed a selective interaction between the alpha-anomer of D-[1-(13)C]glucose and 13C-labels within [13C]tryptophan-labeled GalP, which places the carbon atom at C-1 in the alpha-anomer of D-glucose to within 6 A of the carbonyl carbon of one or more tryptophan residues in the protein. No interaction was detected for the beta-isomer. The role of tryptophan residues in substrate binding was investigated further in CP-MAS experiments to detect D-[1-(13)C]glucose binding to the GalP mutants W371F and W395F before and after the addition of the inhibitor forskolin. The results suggest that both mutants bind D-glucose with similar affinities, but have different affinities for forskolin. This work highlights a useful general experimental strategy for probing the binding sites of membrane proteins, using methodology which overcomes the problems associated with the unfavorable dynamics of weak ligands.

KW - Binding Sites

KW - Biochemistry

KW - Calcium-Binding Proteins

KW - Carbon

KW - Escherichia coli

KW - Forskolin

KW - Glucose

KW - Ligands

KW - Magnetic Resonance Spectroscopy

KW - Mass Spectrometry

KW - Monosaccharide Transport Proteins

KW - Mutation

KW - Periplasmic Binding Proteins

KW - Substrate Specificity

KW - Temperature

KW - Tryptophan

U2 - 10.1021/ja075584k

DO - 10.1021/ja075584k

M3 - Journal article

C2 - 18177040

VL - 130

SP - 1236

EP - 1244

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 4

ER -

Research

Text available via DOI:

Keywords