Weak substrate binding to transport proteins studied by NMR

The weak binding of sugar substrates fails to induce any quantifiable physi cal changes in the L-fucose-H+ symport protein, FucP, from Escherichia coli , and this protein lacks any strongly binding ligands for competitive bindi ng assays. Access to substrate binding behavior is however possible using N MR methods which rely on substrate immobilization for detection. Cross-pola rization from proton to carbon spins could detect the portion of C-13-label ed substrate associated with 0.2 mu mol 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 unlabe led substrates L-fucose and L-galactose but was unaffected by a three- to f ivefold molar excess of the non-transportable stereoisomer D-fucose. FucP a ppeared to bind both anomers of its substrates equally well. An NMR method, designed to measure the rate of substrate exchange, could show that substr ate exchanged slowly with the carrier center (>10(-1) s), although its dyna mics are not necessarily coupled strongly to this site within the protein. Relaxation measurements support this view that fluctuations in the interact ion with substrate would be confined to the binding site in this transport system.