AbstractThe twin-arginine translocase (Tat) is a highly specialised protein transport system, present in prokaryotes and plant chloroplasts. This translocase functions to transport proteins across the cytoplasmic membrane in a fully folded state. Substrates of the Tat system are targeted to the system by N-terminal signal peptides, which bear the consensus motif (S/T)RRxFLK. These proteins often require redox centres for function that must be incorporated prior to transport, with many utilising the molybdenum cofactor Mo-bis-MGD for enzymatic activity during anaerobic respiration. Cofactor loading and protein maturation of these substrates is often coordinated by a ‘Tat proofreading’ process, involving the binding of cytoplasmic chaperone proteins to the substrate signal peptide to prevent premature translocation before maturation is complete. A large group of these proteins form the TorD family of chaperone proteins, of which two examples are DmsD and TorD. DmsD is known to aid maturation of three Tat substrates in Salmonella enterica subsp. enterica serovar Typhimurium; YnfE - the catalytic subunit of selenate reductase, YnfF, and DmsA - the catalytic subunit of dimethyl sulphoxide (DMSO) reductase. TorD assists in the assembly of the catalytic subunit of trimethylamine N-oxide (TMAO) reductase, TorA.
Work presented in this thesis has demonstrated that the interaction between S. Typhimurium DmsD and YnfE requires a hydrophobic stretch of residues on the N-terminal signal peptide of YnfE, the sequence of which is highly conserved amongst signal peptide sequences of all three DmsD target proteins. Genetic and biochemical analysis also revealed residues of importance on the DmsD protein, with a proposed binding mechanism being discussed involving a hydrophobic cleft on the protein surface. The possibility that DmsD is involved in activities other than signal peptide binding was also touched upon. TorA/TorD binding interactions of Escherichia coli were also investigated, and again highlighted the prospect of dual functionality of these chaperone proteins, with TorD amino acid residues being implicated in TorA signal peptide interactions and TMAO reductase activity. High resolution microscopy was employed to enable imaging of TorD within the cellular environment, and super resolution microscopy was utilized to elucidate the interplay between Tat substrates and the Tat translocase. Finally, broad metabolic phenotype screening technology was used to gain an understanding of the broader function of dmsD and other genes in bacterial cell metabolism.
|Date of Award||2014|
|Supervisor||Frank Sargent (Supervisor)|
Investigations into the roles of bacterial TorD family chaperone proteins
Connelly, K. (Author). 2014
Student thesis: Doctoral Thesis › Doctor of Philosophy