Characterization of DmsD - DmsA Twin-Arginine Leader Peptide Binding
Date
2013-05-01
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Abstract
The twin-arginine translocase (Tat) is capable of translocating fully folded, cofactor loaded and often multimeric protein complexes across the inner membrane of bacteria. Many substrates of the Tat system are periplasmic respiratory enzymes that contain a range of redox-active prosthetic groups including molybdenum cofactors. Substrates of the Tat system contain a conserved twin-arginine (RR) motif within the signal sequence at the amino terminus of the pre-protein. System specific chaperones or redox enzyme maturation proteins (REMPs) bind to their cognate twin-arginine leader sequence. The Escherichia coli dimethylsulfoxide reductase complex (DmsABC) is a Tat-dependent enzyme. The twin-arginine leader peptide sequence is located at the amino terminus of the DmsA subunit and has been shown to bind to the DmsD REMP. The research within this thesis focused on characterizing the interaction between the DmsD chaperone and its substrate, the DmsA leader (DmsAL) sequence.
Sequence analysis of homologous DmsD proteins was used to target conserved amino acid residues for mutation and subsequent variant protein production. Twenty-eight single substitution DmsD variants were expressed, purified and assayed for binding to the DmsAL with a high-throughput immunoblot assay. Many of the DmsD residues that were found to reduce binding to DmsAL localized to a pocket on the surface of an E. coli DmsD structure. The DmsAL binding of these DmsD variants was further characterized using isothermal titration calorimetry (ITC) and the multimeric state(s) of each variant was determined using size-exclusion chromatography and native-PAGE analysis.
The DmsAL sequence is composed of four distinct regions. Various portions of the DmsAL peptide were synthesized and ITC was used to investigate binding to DmsD. This was complemented with secondary structure analysis of the DmsAL peptides by circular dichroism spectroscopy. The contiguous hydrophobic region was the smallest portion of the DmsAL peptide able to bind and may form an alpha-helix upon binding to the pocket residues of DmsD.
The studies presented within this thesis reveal details of how DmsD binds to the twin-arginine leader of the Tat-substrate of DMSO reductase through defining residues of the interaction site(s) in both DmsD and the DmsAL and describing the thermodynamic parameters of binding.
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Biochemistry
Citation
Winstone, T. (2013). Characterization of DmsD - DmsA Twin-Arginine Leader Peptide Binding (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/25273