Large Amplitude Twisting Motions of an Interdomain Hinge: A Disulfide Trapping Study of the Galactose-Glucose Binding Protein

Abstract

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.

Dominant role of local dipolar interactions in phosphate binding to a receptor cleft with an electronegative charge surface: Equilibrium, kinetic, and crystallographic studies

“…The Trp would be sensitive to the closure and not the initial phosphate binding to one domain. The study of hinged cleft motion in solution reported here is compatible with that on the periplasmic glucose/galactose-biding protein using disulfide trapping technique (Careaga et al, 1995).…”

Advances in Fluorescence Spectroscopy: Multi-Photon Excitation, Engineered Proteins, Modulation Sensing and Microsecond Rhenium Metal-Ligand Complexes

“…Similar results were obtained with other single cysteine mutants of GGBP [54]. In the absence of glucose, the GGBP is known to become more flexible [55]. It is likely that the flexibility of the protein in the absence of glucose allows ANS to position itself in a more hydrophobic surface of the protein resulting in increased fluorescence intensity.…”

“…This protein binds glucose with a dissociation constant near 0.8 µM [53,54]. The single polypeptide chain of GGBP consists of two domains, the relative positions of which change upon binding of glucose [55] (Fig. 9).…”

Coevolutionary signals across protein lineages help capture multiple protein conformations

“…seems to be functionally relevant because it has been hypothesized to facilitate ribose transfer in the permease complex-a partially closed conformation with a more weakly bound ribose might help in providing an easier release of ribose into the membrane-bound permease (3). A similar twisted intermediate state was suggested for the D-Glucose binding protein, another member of the same family, based on experimental studies using disulfite-trapping and fluorescence spectroscopy (36,37). Very recently, accelerated molecular dynamics were used to suggest the existence of a semiclosed state for the Maltose binding protein, a member of a closely related family (38,39).…”