Dunne, O, Weidenhaupt, M, Callow, P, Martel, A, Moulin, M, Perkins, S, Haertlein, M and Forsyth, VT (2016) Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density culture. European Biophysics Journal, 46 (5). pp. 425-432. ISSN 0175-7571

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Small-angle neutron scattering (SANS) is a powerful technique for the characterisation of macromolecular structures and interactions. Its main advantage over other solution state approaches is the ability to use D2O/H2O solvent contrast variation to selectively match out specific parts of a multi-component system. While proteins, nucleic acids, and lipids are readily distinguished in this way, it is not possible to locate different parts of a protein–protein system without the introduction of additional contrast by selective deuteration. Here, we describe new methods by which ‘matchout labelled’ proteins can be produced using Escherichia coli and Pichia pastoris expression systems in high cell-density cultures. The method is designed to produce protein that has a scattering length density that is very close to that of 100% D2O, providing clear contrast when used with hydrogenated partner proteins in a complex. This allows the production of a single sample system for which SANS measurements at different solvent contrasts can be used to distinguish and model the hydrogenated component, the deuterated component, and the whole complex. The approach, which has significant cost advantages, has been extensively tested for both types of expression system.

Item Type: Article
Additional Information: This is the final published version of the article (version of record). It first appeared online via Springer Verlag at http://link.springer.com/article/10.1007/s00249-016-1186-2 - please refer to any applicable terms of use of the publisher.
Uncontrolled Keywords: matchout deuteration, contrast variation, sans, neutron scattering
Divisions: Faculty of Natural Sciences > School of Chemical and Physical Sciences
Depositing User: Symplectic
Date Deposited: 31 Jan 2019 09:47
Last Modified: 10 Jan 2020 13:22
URI: https://eprints.keele.ac.uk/id/eprint/5738

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