Tritium NMR spectroscopy of ligand binding to maltose-binding protein.
Tritium-labeled alpha- and beta-maltodextrins have been used to study their complexes with maltose-binding protein (MBP), a 40-kDa bacterial protein. Five substrates, from maltose to maltohexaose, were labeled at their reducing ends and their binding studied. Tritium NMR spectroscopy of the labeled sugars showed large upfield chemical shift changes upon binding and strong anomeric specificity. At 10 degrees C, MBP bound alpha-maltose with 2.7 +/- 0.5-fold higher affinity than beta-maltose, and, for longer maltodextrins, the ratio of affinities (KD beta/KD alpha) was even larger (between 10 and 30). The maximum chemical shift change was 2.2 ppm, suggesting that the reducing end of bound alpha-maltodextrin makes close contact with an aromatic residue in the MBP-binding site. Experiments with maltotriose (and longer maltodextrins) also revealed the presence of two bound beta-maltotriose resonances in rapid exchange. We interpret these two resonances as arising from two distinct sugar-protein complexes. In one complex, the beta-maltodextrin is bound by its reducing end, and, in the other complex, the beta-maltodextrin is bound by the middle glucose residue(s). This interpretation also suggests how MBP is able to bind both linear and circular maltodextrins.
Gehring
K
K
Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Williams
P G
PG
Pelton
J G
JG
Morimoto
H
H
Wemmer
D E
DE
D018528:ATP-Binding Cassette Transporters; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis; D029968:Escherichia coli Proteins; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008320:Maltose / Q000737:chemistry; D058619:Maltose-Binding Proteins; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D011134:Polysaccharides / Q000378:metabolism*; D011487:Protein Conformation; D014316:Tritium
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011134:Polysaccharides; C461599:maltose transport system, E coli; D014316:Tritium; D008320:Maltose; C008315:maltodextrin