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

Dow, J.M., Grahl, S., Ward, R., Evans, R., Byron, O. , Norman, D.G., Palmer, T. and Sargent, F. (2014) Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone. FEBS Journal, 281(1), pp. 246-260. (doi:10.1111/febs.12592)

Dow, J.M., Grahl, S., Ward, R., Evans, R., Byron, O. , Norman, D.G., Palmer, T. and Sargent, F. (2014) Characterization of a periplasmic nitrate reductase in complex with its biosynthetic chaperone. FEBS Journal, 281(1), pp. 246-260. (doi:10.1111/febs.12592)

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Abstract

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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Byron, Professor Olwyn
Authors: Dow, J.M., Grahl, S., Ward, R., Evans, R., Byron, O., Norman, D.G., Palmer, T., and Sargent, F.
College/School:College of Medical Veterinary and Life Sciences > School of Life Sciences
Journal Name:FEBS Journal
Publisher:Wiley
ISSN:1742-464X
ISSN (Online):1742-4658
Copyright Holders:Copyright © 2014 The Authors
First Published:First published in FEBS Journal 281(1):246-260
Publisher Policy:Reproduced under a Creative Commons License

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