Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 1998 Dec;75(6):2794–2800. doi: 10.1016/S0006-3495(98)77722-7

Weak substrate binding to transport proteins studied by NMR.

P J Spooner 1, W J O'Reilly 1, S W Homans 1, N G Rutherford 1, P J Henderson 1, A Watts 1
PMCID: PMC1299952  PMID: 9826601

Abstract

The weak binding of sugar substrates fails to induce any quantifiable physical changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli, and this protein lacks any strongly binding ligands for competitive binding assays. Access to substrate binding behavior is however possible using NMR methods which rely on substrate immobiliza-tion for detection. Cross-polarization from proton to carbon spins could detect the portion of 13C-labeled substrate associated with 0.2 micromol of the functional transport system overexpressed in the native membranes. The detected substrate was shown to be in the FucP binding site because its signal was diminished by the unlabeled substrates L-fucose and L-galactose but was unaffected by a three- to fivefold molar excess of the non-transportable stereoisomer D-fucose. FucP appeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substrate exchanged slowly with the carrier center (>10(-1) s), although its dynamics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interaction with substrate would be confined to the binding site in this transport system.

Full Text

The Full Text of this article is available as a PDF (95.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradley S. A., Tinsley C. R., Muiry J. A., Henderson P. J. Proton-linked L-fucose transport in Escherichia coli. Biochem J. 1987 Dec 1;248(2):495–500. doi: 10.1042/bj2480495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Cornell B. A., Hiller R. G., Raison J., Separovic F., Smith R., Vary J. C., Morris C. Biological membranes are rich in low-frequency motion. Biochim Biophys Acta. 1983 Jul 27;732(2):473–478. doi: 10.1016/0005-2736(83)90065-2. [DOI] [PubMed] [Google Scholar]
  3. Derrick J. P., Lian L. Y., Roberts G. C., Shaw W. V. Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited 1H NMR and site-directed mutagenesis. Biochemistry. 1992 Sep 8;31(35):8191–8195. doi: 10.1021/bi00150a010. [DOI] [PubMed] [Google Scholar]
  4. Gunn F. J., Tate C. G., Henderson P. J. Identification of a novel sugar-H+ symport protein, FucP, for transport of L-fucose into Escherichia coli. Mol Microbiol. 1994 Jun;12(5):799–809. doi: 10.1111/j.1365-2958.1994.tb01066.x. [DOI] [PubMed] [Google Scholar]
  5. Gunn F. J., Tate C. G., Sansom C. E., Henderson P. J. Topological analyses of the L-fucose-H+ symport protein, FucP, from Escherichia coli. Mol Microbiol. 1995 Feb;15(4):771–783. doi: 10.1111/j.1365-2958.1995.tb02384.x. [DOI] [PubMed] [Google Scholar]
  6. Henderson P. J., Macpherson A. J. Assay, genetics, proteins, and reconstitution of proton-linked galactose, arabinose, and xylose transport systems of Escherichia coli. Methods Enzymol. 1986;125:387–429. doi: 10.1016/s0076-6879(86)25033-8. [DOI] [PubMed] [Google Scholar]
  7. Henderson P. J. The 12-transmembrane helix transporters. Curr Opin Cell Biol. 1993 Aug;5(4):708–721. doi: 10.1016/0955-0674(93)90144-f. [DOI] [PubMed] [Google Scholar]
  8. Lian L. Y., Barsukov I. L., Sutcliffe M. J., Sze K. H., Roberts G. C. Protein-ligand interactions: exchange processes and determination of ligand conformation and protein-ligand contacts. Methods Enzymol. 1994;239:657–700. doi: 10.1016/s0076-6879(94)39025-8. [DOI] [PubMed] [Google Scholar]
  9. Loo D. D., Zeuthen T., Chandy G., Wright E. M. Cotransport of water by the Na+/glucose cotransporter. Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):13367–13370. doi: 10.1073/pnas.93.23.13367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mitchell P. Osmochemistry of solute translocation. Res Microbiol. 1990 Mar-Apr;141(3):286–289. doi: 10.1016/0923-2508(90)90002-8. [DOI] [PubMed] [Google Scholar]
  11. Muiry J. A., Gunn T. C., McDonald T. P., Bradley S. A., Tate C. G., Henderson P. J. Proton-linked L-rhamnose transport, and its comparison with L-fucose transport in Enterobacteriaceae. Biochem J. 1993 Mar 15;290(Pt 3):833–842. doi: 10.1042/bj2900833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  13. Spooner P. J., Duralski A. A., Rankin S. E., Pinheiro T. J., Watts A. Dynamics in a protein-lipid complex: nuclear magnetic resonance measurements on the headgroup of cardiolipin when bound to cytochrome c. Biophys J. 1993 Jul;65(1):106–112. doi: 10.1016/S0006-3495(93)81048-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Spooner P. J., Rutherford N. G., Watts A., Henderson P. J. NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3877–3881. doi: 10.1073/pnas.91.9.3877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Walmsley A. R., Martin G. E., Henderson P. J. 8-Anilino-1-naphthalenesulfonate is a fluorescent probe of conformational changes in the D-galactose-H+ symport protein of Escherichia coli. J Biol Chem. 1994 Jun 24;269(25):17009–17019. [PubMed] [Google Scholar]
  16. Watts A., Ulrich A. S., Middleton D. A. Membrane protein structure: the contribution and potential of novel solid state NMR approaches. Mol Membr Biol. 1995 Jul-Sep;12(3):233–246. doi: 10.3109/09687689509072423. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES