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Lookup NU author(s): Professor Tracy Palmer FRS FRSE FMedSciORCiD, Professor Frank SargentORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
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. © 2013 The Authors.
Author(s): Dow JM, Grahl S, Ward R, Evans R, Byron O, Norman DG, Palmer T, Sargent F
Publication type: Article
Publication status: Published
Journal: FEBS Journal
Year: 2014
Volume: 281
Issue: 1
Pages: 246-260
Print publication date: 01/01/2014
Online publication date: 04/11/2013
Acceptance date: 28/10/2013
Date deposited: 15/02/2019
ISSN (print): 1742-464X
ISSN (electronic): 1742-4658
Publisher: Wiley-Blackwell Publishing Ltd.
URL: https://doi.org/10.1111/febs.12592
DOI: 10.1111/febs.12592
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