Tat signal peptide recognition during protein maturation and export

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.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Cyanophage MazG is a pyrophosphohydrolase but unable to hydrolyse magic spot nucleotides

Bacteriophage possess a variety of auxiliary metabolic genes (AMGs) of bacterial origin. These proteins enable them to maximise infection efficiency, subverting bacterial metabolic processes for the purpose of viral genome replication and synthesis of the next generation of virion progeny. Here, we examined the enzymatic activity of a cyanophage MazG protein – a putative pyrophosphohydrolase previously implicated in regulation of the stringent response via reducing levels of the central alarmone molecule (p)ppGpp. We demonstrate however, that the purified viral MazG shows no binding or hydrolysis activity against (p)ppGpp. Instead, dGTP and dCTP appear to be the preferred substrates of this protein, consistent with a role preferentially hydrolysing deoxyribonucleotides from the high GC content host Synechococcus genome. This showcases a new example of the fine‐tuned nature of viral metabolic processes

Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone

Escherichia coliis a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase, NapA, contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD.In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1:1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapAwas at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken altogether, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor

Oep23 forms an ion channel in the chloroplast outer envelope

Background Metabolite, ion and protein translocation into chloroplasts occurs across two membranes, the inner and the outer envelope. Solute and metabolite channels fulfill very important functions in integrating the organelles into the metabolic network of the cell. However so far only a few have been identified. Here we describe the identification and the characterization of the outer envelope protein of 23 kDa, Oep23 from garden pea. Results Oep23 is found in the entire plant lineage from green algae to flowering plants. It is expressed in all organs and developmental states tested so far. The reconstituted recombinant protein Oep23 from pea forms a high conductance ion channel with a maximal conductance in the fully open state of 466 ± 14pS at a holding potential of +100 mV (in 250 mM KCl). The Oep23 channel is cation selective (PK+ : PCl- = 15 : 1) with a voltage dependent open probability of maximal Vmem = 0 mV. Conclusion The data indicate that the Oep23 activity represents a single channel unit and does not assemble into a multiple pore complex like bacterial type porins or mitochondrial voltage dependent anion channel. Thus, Oep23 represents a new member of ion channels in the outer envelope of chloroplasts involved in solute exchange

Oep23 forms an ion channel in the chloroplast outer envelope

Background Metabolite, ion and protein translocation into chloroplasts occurs across two membranes, the inner and the outer envelope. Solute and metabolite channels fulfill very important functions in integrating the organelles into the metabolic network of the cell. However so far only a few have been identified. Here we describe the identification and the characterization of the outer envelope protein of 23 kDa, Oep23 from garden pea. Results Oep23 is found in the entire plant lineage from green algae to flowering plants. It is expressed in all organs and developmental states tested so far. The reconstituted recombinant protein Oep23 from pea forms a high conductance ion channel with a maximal conductance in the fully open state of 466 ± 14pS at a holding potential of +100 mV (in 250 mM KCl). The Oep23 channel is cation selective (PK+ : PCl- = 15 : 1) with a voltage dependent open probability of maximal Vmem = 0 mV. Conclusion The data indicate that the Oep23 activity represents a single channel unit and does not assemble into a multiple pore complex like bacterial type porins or mitochondrial voltage dependent anion channel. Thus, Oep23 represents a new member of ion channels in the outer envelope of chloroplasts involved in solute exchange

Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide

The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK twin-arginine amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a a-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway

Cysteine Scanning Mutagenesis and Topological Mapping of the Escherichia coli Twin-Arginine Translocase TatC Component▿

The TatC protein is an essential component of the Escherichia coli twin-arginine (Tat) protein translocation pathway. It is a polytopic membrane protein that forms a complex with TatB, together acting as the receptor for Tat substrates. In this study we have constructed 57 individual cysteine substitutions throughout the protein. Each of the substitutions resulted in a TatC protein that was competent to support Tat-dependent protein translocation. Accessibility studies with membrane-permeant and -impermeant thiol-reactive reagents demonstrated that TatC has six transmembrane helices, rather than the four suggested by a previous study (K. Gouffi, C.-L. Santini, and L.-F. Wu, FEBS Lett. 525:65-70, 2002). Disulfide cross-linking experiments with TatC proteins containing single cysteine residues showed that each transmembrane domain of TatC was able to interact with the same domain from a neighboring TatC protein. Surprisingly, only three of these cysteine variants retained the ability to cross-link at low temperatures. These results are consistent with the likelihood that most of the disulfide cross-links are between TatC proteins in separate TatBC complexes, suggesting that TatC is located on the periphery of the complex

Characterization of the periplasmatic nitrate reductase in complex with its biosynthetic chaperone

Escherichia coli is a Gram-negative bacterium that can use nitrate during anaerobic respiration. The catalytic subunit of the periplasmic nitrate reductase NapA contains two types of redox cofactor and is exported across the cytoplasmic membrane by the twin-arginine protein transport pathway. NapD is a small cytoplasmic protein that is essential for the activity of the periplasmic nitrate reductase and binds tightly to the twin-arginine signal peptide of NapA. Here we show, using spin labelling and EPR, that the isolated twin-arginine signal peptide of NapA is structured in its unbound form and undergoes a small but significant conformational change upon interaction with NapD. In addition, a complex comprising the full-length NapA protein and NapD could be isolated by engineering an affinity tag onto NapD only. Analytical ultracentrifugation demonstrated that the two proteins in the NapDA complex were present in a 1 : 1 molar ratio, and small angle X-ray scattering analysis of the complex indicated that NapA was at least partially folded when bound by its NapD partner. A NapDA complex could not be isolated in the absence of the NapA Tat signal peptide. Taken together, this work indicates that the NapD chaperone binds primarily at the NapA signal peptide in this system and points towards a role for NapD in the insertion of the molybdenum cofactor

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