Thermodynamic and Hydrodynamic Characterization of the Interaction Between DmsD and the DmsA Twin-Arginine Leader Peptide

Abstract

The system specific chaperone DmsD plays a role in the maturation of the DMSO reductase enzyme through its interaction with the twin-arginine leader peptide of the catalytic subunit DmsA, prior to its assembly into the holo-enzyme. A pocket of residues, clustered together within the structure of DmsD, has previously been shown to be important for binding a fusion protein composed of 43 of the 45 amino acid residues of the DmsA leader peptide however the region of the DmsA leader peptide that interacts with DmsD has not been identified. Various portions of the DmsA leader peptide were synthesized and assayed for binding to DmsD using isothermal titration calorimetry. A peptide composed of 27 amino acid residues near the C-terminus of the DmsA leader sequence was found to bind to DmsD and subsequently used to characterize the thermodynamics of binding of each of the DmsD variant proteins previously shown to be important for binding to the DmsA leader peptide. Size exclusion chromatography and native-PAGE were used to determine the effect of peptide binding on multimeric state and electrophoretic mobility for each of the variant proteins. In the presence of the peptide, wild type DmsD migrates faster on native-PAGE but remains monomeric while some DmsD variant proteins undergo oligomerization while still changing to a the same faster migrating form on native-PAGE. The electrophoretic mobility, multimeric forms and thermodynamics of peptide binding of mutations in the chaperone leader-binding site are compared.