The twin-arginine (Tat) system is a specialised translocation machine found in prokaryotes and chloroplasts which serves to export fully folded proteins across the cytoplasmic membrane. Proteins are specifically targeted to this system by N-terminal signal peptides which bear a conserved SRRxFLK motif. Tat dependent proteins often contain redox factors that must be inserted prior to translocation. The E. coli Tat substrates trimethylamine Noxide
reductase (Tor) and periplasmic nitrate reductase (Nap) are two such substrates
which require the molybdenum cofactor Mo-bis-MGD in order to function as anaerobic reductases.
Pre-export assembly of Tor and Nap are subject to a process called Tat proofreading, where the substrate specific chaperones, TorD and NapD, bind tightly to the signal peptides of these substrates, TorA and NapA, respectively, assisting in the assembly of the proteins.
Work described in this thesis demonstrates that TorD simultaneously binds to two independent sites present on the TorA, one present in the mature domain of the substrate and one in the signal peptide. Biophysical and structural analysis shows that the pre-export complexes of TorD and TorA appear identical regardless of whether the TorA signal peptide is present or absent. However, the complex purified in the absence of the TorA signal
peptide is able to interact with extrinsically-added signal peptide, confirming the presence of a separate signal peptide binding site. By contrast to TorD, NapD was shown to bind exclusively to the signal peptide of its substrate, NapA. Pre-export complexes of TorAD and NapDA showed similar low resolution structures, with both NapA and TorA exhibiting flexibility in Domain IV. It is hypothesised that this Domain acts as a gate, allowing access of
the cofactor, and that closing of this gate is the final step in the folding of the substrate.
|Date of Award||2013|
|Supervisor||Tracy Palmer (Supervisor)|