Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578817
Title: Tat signal peptide recognition during protein maturation and export
Author: Grahl, Sabine
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2011
Availability of Full Text:
Access through EThOS:
Access through Institution:
Abstract:
Nitrogen is one of the most abundant elements on Earth and mostly found in the atmosphere as the inert gas N2. Therefore the nitrogen cycle is important for maintaining the bioavailabilty of nitrogen for organisms. Denitrification is a process that closes the nitrogen cycle by subsequent conversion of nitrate to dinitrogen with reduction of nitrate to nitrite being the very first necessary step. The bacterial periplasmic nitrate reductase NapA is one of those nitrate reducing enzymes and contains a molybdenum cofactor and a [4Fe-4S] cluster as cofactors. As a periplasmic terminal reductase NapA has an N-terminal signal peptide harbouring a Tat (twin-arginine translocation) motif, which follows closely the consensus S/T-R-R-x-F-L-K. As with other proteins transported via the Tat pathway, NapA needs to be fully folded, and cofactor insertion needs to be completed, prior to export. This is assured by an individual chaperone in a process called ‘Tat-proofreading’. The proofreading chaperone for NapA is NapD, which had been previously shown to interact tightly with the signal peptide of NapA.In this work the binding epitope on the Escherichia coli NapA signal peptide recognised by NapD was mapped for the first time. The key amino acid residues (NapA R6, K10, A17) overlapped with the Tat targeting motif and were further characterized in vitro and in vivo for their importance in NapD binding, Tat transport and NapA biosynthesis. In addition, napD suppressor mutants able to re-bind the NapA A17Q variant were isolated. NMR spectroscopy revealed the 3D solution structure of NapD in complex with the NapA signal peptide. Interestingly, the signal peptide of NapA is a-helical when bound to NapD. Overall, the structure supports strongly that NapA residues R6, K10 and A17 interact with NapD. Pulsed EPR spectroscopy on the isolated signal peptide indicated structural changes of the NapA signal peptide between NapD bound and unbound states, though it was believed that an overall a-helical structure was maintained. Co-purification studies of the complete NapDA complex for crystallisation trials resulted in increased information on the behaviour of the complex and the order of cofactor insertion into NapA. Finally, an in vitro translation and cross-linking approach was attempted with the aim of addressing whether direct contact was made between NapD and the Tat translocase. In addition, functional chromosomal fusions of either NapD or NapA with fluorescent proteins were generated to form a basis for a future project based on fluorescence correlation spectroscopy in living cells. This project has therefore provided fresh insight into the NapA-NapD interaction at the molecular level and laid the foundations for future research in this area.
Supervisor: Sargent, Frank Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.578817  DOI: Not available
Share: