Open conformation of a substrate-binding cleft: 19F NMR studies of cleft angle in the D-galactose chemosensory receptor.
The Escherichia coli D-galactose and D-glucose receptor is a two-domain structure with a sugar-binding site at the interface between domains. The structure of the closed cleft containing bound D-glucose has been determined crystallographically, but the open cleft remains to be characterized. The present study illustrates a generalizable approach that is used to detect and analyze both the open- and closed-cleft conformations in solution. A 19F nucleus located inside the cleft is monitored by 19F NMR. When the cleft is occupied by D-glucose, the 19F nucleus is found to be inaccessible to the aqueous paramagnetic probe Gd-EDTA, verifying that the occupied cleft is closed in solution and inaccessible to bulk solvent. When the cleft is empty, the 19F nucleus becomes accessible to the paramagnet such that the distance of closest approach is r less than or equal to 10 A, indicating that the empty cleft opens at least transiently by an angle theta greater than or equal to 18 +/- 3 degrees.
Luck
L A
LA
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Falke
J J
JJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D003196:Computer Graphics; D004492:Edetic Acid / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D005461:Fluorine; D005682:Gadolinium; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D009942:Organometallic Compounds / Q000737:chemistry; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D014364:Tryptophan
D009942:Organometallic Compounds; D011956:Receptors, Cell Surface; C039707:galactose receptor; C027786:glucose receptor; C041365:gadolinium EDTA; D005461:Fluorine; D014364:Tryptophan; D004492:Edetic Acid; D005682:Gadolinium; D005947:Glucose; D005690:Galactose