Weak substrate binding to transport proteins studied by NMR.
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.
Spooner
P J
PJ
Biomembrane Structure Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom. spooner@bioch.ox.ac.uk
O'Reilly
W J
WJ
Homans
S W
SW
Rutherford
N G
NG
Henderson
P J
PJ
Watts
A
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D055592:Biophysical Phenomena; D001703:Biophysics; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005643:Fucose / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011485:Protein Binding; D027981:Symporters; D013816:Thermodynamics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C088688:FucP protein, E coli; D008024:Ligands; D027981:Symporters; D005643:Fucose