Large amplitude twisting motions of an interdomain hinge: a disulfide trapping study of the galactose-glucose binding protein.
The galactose--glucose binding protein possesses two structural domains bordering a ligand binding cleft, with three polypeptide strands serving as a flexible hinge connecting the two domains. The hinge is known to bend, enabling the cleft to open by an angle of at least 18 degrees. Here the twisting motions of the hinge were examined by placing pairs of engineered cysteines on the perimeter of the cleft to generate six stable di-cysteine proteins. Each cysteine pair introduced reactive sulfhydryls into both rims of the cleft, one in the N-terminal domain and the other in the C-terminal domain. Collisions between sulfhydryls in different domains were trapped by disulfide formation, yielding sensitive detection of large amplitude domain rotations. When the cleft was occupied by the ligand D-glucose, counterclockwise hinge twist rotations were detected with amplitudes up to 36 degrees, and frequencies ranging from 10(1) to 10(3) collisions s-1. Removal of ligand from the cleft increased the range of twist angles 3-fold and the frequency of motions up to 10(2)-fold. Thus, in this representative hinged cleft protein, large amplitude hinge twist motions occur on biologically relevant timescales. The functional implications of such motions are discussed.
Careaga
C L
CL
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Sutherland
J
J
Sabeti
J
J
Falke
J J
JJ
D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009038:Motion; D033902:Periplasmic Binding Proteins; D015202:Protein Engineering
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004220:Disulfides; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein