title: Influence of GTP on system specific chaperone - Twin arginine signal peptide interaction.
abstract: Many bacterial respiratory redox enzymes depend on the twin-arginine translocase (Tat) system for translocation and membrane insertion. Tat substrates contain an N-terminal twin-arginine (SRRxFLK) motif serving as the targeting signal towards the translocon. Many Tat substrates have a system specific chaperone - redox enzyme maturation protein (REMP) - for final folding and assembly prior to Tat binding. The REMP DmsD strongly interacts with the twin-arginine motif of the DmsA signal sequence of dimethyl sulfoxide (DMSO) reductase. In this study, we have utilized the in vitro protein-protein interaction technique of an affinity pull down assay, as well as protein thermal stability measurement via differential scanning fluorimetry (DSF) to investigate the interaction of guanosine nucleotides (GNPs) with DmsD. Here we have shown highly cooperative binding of DmsD with GTP. A dissociative ligand-binding style isotherm was generated upon GTP titration into the DmsD:DmsAL interaction, yielding sigmoidal release of DmsD with a Hill coefficient of 2.09 and a dissociation constant of 0.99 mM. DSF further illustrated the change in thermal stability upon DmsD interaction with DmsAL and GTP. These results imply the possibility of DmsD detection and binding of GTP during the DMSO protein maturation mechanism, from ribosomal translation to membrane targeting and final assembly. Conceivably, GTP is shown to act as a molecular regulator in the biochemical pathway.
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada.
authors: Stephana J
authors: SJ
authors: Cherak
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada. Electronic address: turnerr@ucalgary.ca.
authors: Raymond J
authors: RJ
authors: Turner
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione; D006160:Guanosine Triphosphate / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D055550:Protein Stability; D012685:Sepharose / Q000031:analogs & derivatives; D000069294:Twin-Arginine-Translocation System / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C434268:DmsD protein, E coli; D029968:Escherichia coli Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007506:Iron-Sulfur Proteins; D018832:Molecular Chaperones; D021382:Protein Sorting Signals; D000069294:Twin-Arginine-Translocation System; D006160:Guanosine Triphosphate; D012685:Sepharose; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase; D005978:Glutathione
keywords: Binding; Differential scanning fluorimetry; Dissociation; DmsA signal peptide; DmsD; Guanosine nucleotides; Twin-arginine translocase