Dunne, O and Weidenhaupt, M and Callow, P and Martel, A and Moulin, M and Perkins, SJ and 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 cultures. European Biophysics Journal. ISSN 1432-1017

[img]
Preview
Text
V Forsyth - Matchout deuterium labelling of proteins for small-angle neutron scattering studies....pdf - Published Version
Available under License Creative Commons Attribution.

Download (1MB) | Preview

Abstract

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
Uncontrolled Keywords: matchout deuteration; contrast variation; SANS; Neutron scattering
Subjects: ?? Contrast variation ??
?? Matchout deuteration ??
?? Neutron scattering ??
Q Science > QD Chemistry
?? SANS ??
Divisions: Faculty of Natural Sciences > School of Physical and Geographical Sciences
Related URLs:
Depositing User: Symplectic
Date Deposited: 08 Dec 2016 15:49
Last Modified: 08 Dec 2016 15:49
URI: http://eprints.keele.ac.uk/id/eprint/2584

Actions (login required)

View Item View Item