The D-galactose chemosensory receptor of Escherichia coli is a .32 kDa globular protein possessing two distinct structural domains, each organized in an alpha/beta folding motif. Helices I and X lie at adjacent approximately parallel positions on the surface of the N-terminal domain, near the hinge region. In order to analyze the relative thermal motions of these two helices, the present study utilizes a generalizable disulfide trapping approach: first, site-directed mutagenesis is used to place a pair of cysteine residues at locations of interest on the protein surface, then disulfide bond formation is used to trap intramolecular cysteine-cysteine collisions resulting from thermal motions. Specifically, four engineered di-cysteine receptors have been constructed, each possessing one cysteine at position 26 on helix I, and a second cysteine at varying positions on helix X. A fifth control receptor possesses one cysteine at position 26, and a second on the opposite surface of the molecule. These surface cysteine substitutions have little or no effect on the measurable receptor parameters as judged by ligand binding equilibria and kinetics, protein stability, and 19F nuclear magnetic resonance, indicating that the engineered receptors are useful probes of native backbone dynamics. Spatial and kinetic features of backbone motions have been investigated by measuring intramolecular disulfide formation rates for cysteine pairs in the fully liganded receptor. The resulting rates decrease monotonically with increasing distance between cysteines in the crystal structure, while no disulfide formation is observed for the control pair unless the molecule is unfolded. The minimum translational amplitudes of the observed backbone motions range from 4.5 to 15.2 A, and the minimum rotational amplitudes are as large as 35 degrees. For each motion the rate of intramolecular sulfhydryl-sulfhydryl collision has been estimated from the measured rate of disulfide formation: the 4.5 and 15.2 A translations yield approximately 10(4) and approximately 10 collisions s-1 molecule-1, respectively. These collision rates, which are faster than ligand dissociation, likely underestimate the actual motional frequencies since only an undetermined fraction of the total motions yield collisions. The simplest plausible trajectory capable of producing such collisions is a rate-limiting translation of one or both helices along their long axes, coupled with minor helix rotations. When sugar is removed from the receptor, a substantial increase in backbone dynamics is observed, indicating the presence of new long-range backbone trajectories. Overall, the results suggest that internal motions in proteins may have larger amplitudes than previously observed.

Cysteine residues introduced by site-directed mutagenesis have been used to probe the conformation and dynamics of two receptors in the E. coli chemotaxis pathway. (a) Thermal motions of the polypeptide backbone were investigated in the periplasmic D-galactose and D-glucose receptor, a globular protein of known structure. Disulfide bond formation between pairs of engineered sulfhydryls were used to trap collisions during the relative motions of surface alpha-helices I and X. Motions with amplitudes ranging from 4.5 to 15.2 A were detected on timescales ranging from 10(-4) to 10(-1) s, respectively. These results suggest that thermal backbone motions may have larger amplitudes than previously thought. (b) Conformational features of the transmembrane aspartate transducer have been investigated. Engineered sulfhydryls were used to ascertain the location and orientations of two putative transmembrane alpha-helices in the primary structure, to investigate the packing of these helices, to determine the oligomer and surface structures, and to detect thermal and ligand-induced dynamics of the polypeptide backbone. A model for the folded conformation of the transducer oligomer is reviewed.

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.