title: The mechanism of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus.
abstract: The kinetics of ligand binding to the periplasmic C4-dicarboxylate binding protein (DctP) from Rhodobacter capsulatus were investigated by exploiting the changes in the intrinsic fluorescence of the protein upon binding ligands. Steady state measurements have shown that L-malate, succinate, and fumarate are all bound with sub-micromolar Kd values, whereas D-malate is bound 2 orders of magnitude more weakly. Stopped-flow studies have revealed that the binding process involves at least three steps. In the absence of ligand, the protein is in equilibrium between an essentially nonbinding form, BP1, and the binding form, BP2. Ligands bind to the BP2 form, shifting the equilibrium toward the BP2-L conformation, and also inducing a further isomerization of the protein, to the BP3-L form. The kinetic properties of the four different conformational states of the DctP protein identified in this study would be consistent with their identification as the closed-conformation, the open-conformation, an open-liganded conformation, and a closed-liganded conformation. The latter three states have been identified by x-ray crystallographic studies of binding proteins, but no kinetic or structural data have been presented previously to support the possibility of a closed but unliganded conformation.
authors.affiliation: Krebs and Robert Hill Institutes, Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.
authors.forename: A R
authors.initials: AR
authors.lastname: Walmsley
authors.forename: J G
authors.initials: JG
authors.lastname: Shaw
authors.forename: D J
authors.initials: DJ
authors.lastname: Kelly
mesh_terms: D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D029382:Dicarboxylic Acid Transporters; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters; D008024:Ligands; C074174:DctP protein, Rhodobacter capsulatus