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Related Experiment Videos

Weak substrate binding to transport proteins studied by NMR

P J Spooner1, W J O'Reilly, S W Homans

  • 1Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom. spooner@bioch.ox.ac.uk

Biophysical Journal
|November 25, 1998
PubMed
Summary

Researchers used NMR to study the L-fucose-H+ symporter (FucP) in E. coli. They found that while substrate binding is weak, it occurs specifically within the FucP binding site with slow exchange rates.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • The L-fucose-H+ symporter (FucP) from Escherichia coli facilitates the transport of L-fucose across the cell membrane.
  • Understanding the substrate binding and transport mechanism of FucP is crucial for elucidating sugar symporter function.
  • Direct observation of weak substrate interactions has been challenging due to the lack of significant conformational changes.

Purpose of the Study:

  • To investigate the substrate binding interactions of the L-fucose-H+ symporter (FucP) from Escherichia coli.
  • To characterize the binding site and dynamics of substrate interaction using biophysical methods.
  • To determine the affinity and specificity of FucP for its substrate.

Main Methods:

  • Utilized Nuclear Magnetic Resonance (NMR) spectroscopy, specifically cross-polarization from proton to carbon spins, for detecting bound substrate.

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  • Employed competitive binding assays with labeled and unlabeled substrates (L-fucose, L-galactose, D-fucose) to confirm binding site specificity.
  • Applied NMR-based methods to measure the rate of substrate exchange and relaxation measurements to probe binding site dynamics.

    • Main Results:

    • NMR detected 13C-labeled L-fucose bound to overexpressed FucP in native membranes, indicating substrate association with the transport system.
    • Binding was confirmed to be specific to the FucP binding site, as evidenced by competition with L-fucose and L-galactose, but not D-fucose.
    • FucP binds both anomers of its substrate equally and exhibits slow substrate exchange rates (>10(-1) s) with the carrier.
    • Relaxation measurements suggest that substrate-binding fluctuations are localized within the binding site.

    Conclusions:

    • Despite weak binding and lack of detectable conformational changes, NMR methods successfully characterized substrate interaction with FucP.
    • The study confirms the specific binding of L-fucose to the FucP transporter and provides insights into the slow dynamics of substrate exchange.
    • Findings contribute to the understanding of sugar-H+ symporter mechanisms at a molecular level.