Perturbation of the equilibrium between open and closed conformations of the periplasmic C4-dicarboxylate binding protein from Rhodobacter capsulatus.
A kinetic and thermodynamic analysis has been carried out on the conformational transitions of the periplasmic C4-dicarboxylate binding protein (DctP) from the photosynthetic bacterium Rhodobacter capsulatus. This protein is distinct from other periplasmic binding proteins characterized to date in that the transition between the putative closed-unliganded (BP1) and open-unliganded (BP2) conformations is slow compared to the rate of ligand binding [Walmsley, A. R., Shaw, J. G., & Kelly, D. J. (1992) J. Biol. Chem. 276, 8064-8072]. Using stopped-flow fluorescence techniques, we have probed the conformational dynamics of the closed to open transition of DctP in the absence and presence of ligand. Both the forward rate constant for the BP1 to BP2 interconversion (k1) and the fumarate dissociation rate constant (k-3) were found to increase in a biphasic manner between pH 5 and pH 11. The data were fitted to a two-pKa function which gave pKa values of 10.3 and 5.4 for the BP1 to BP2 interconversion and 8.9 and 4.5 for the closed-liganded (BP3L) to open-liganded (BP2L) transition. An increase in ionic strength at constant pH resulted in a hyperbolic increase in both k1 and k-3 to maximal rates that were similar in each case to the values obtained in pH variation experiments. Measurement of the temperature dependencies of k1 and k-3 also gave similar activation energies. Gibbs free energy, enthalpy, and entropy changes were determined for the open to closed transitions of DctP in both the presence and absence of ligand.(ABSTRACT TRUNCATED AT 250 WORDS)
Walmsley
A R
AR
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, United Kingdom.
Shaw
J G
JG
Kelly
D J
DJ
D002352:Carrier Proteins / Q000737:chemistry*; D029382:Dicarboxylic Acid Transporters; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D002352:Carrier Proteins; D029382:Dicarboxylic Acid Transporters