title: Matchout deuterium labelling of proteins for small-angle neutron scattering studies using prokaryotic and eukaryotic expression systems and high cell-density cultures.
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.
authors.affiliation: Department of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.|Institut Laue Langevin, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France.
authors.forename: O
authors.initials: O
authors.lastname: Dunne
authors.affiliation: University Grenoble Alpes, CNRS, LMGP, F-38000, Grenoble, France.
authors.forename: M
authors.initials: M
authors.lastname: Weidenhaupt
authors.affiliation: Institut Laue Langevin, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France.
authors.forename: P
authors.initials: P
authors.lastname: Callow
authors.affiliation: Institut Laue Langevin, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France.
authors.forename: A
authors.initials: A
authors.lastname: Martel
authors.affiliation: Institut Laue Langevin, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France.
authors.forename: M
authors.initials: M
authors.lastname: Moulin
authors.affiliation: Department of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: S J
authors.initials: SJ
authors.lastname: Perkins
authors.affiliation: Institut Laue Langevin, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France.
authors.forename: M
authors.initials: M
authors.lastname: Haertlein
authors.affiliation: Institut Laue Langevin, 71 avenue des Martyrs, 38042, Grenoble Cedex 9, France. tforsyth@ill.eu.|Macromolecular Structure Group, Faculty of Natural Sciences, Keele University, Staffordshire, ST5 5BG, UK. tforsyth@ill.eu.
authors.forename: V T
authors.initials: VT
authors.lastname: Forsyth
mesh_terms: D002452:Cell Count; D002478:Cells, Cultured; D017242:Complement Factor H / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003903:Deuterium / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D007553:Isotope Labeling; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D033363:Neutron Diffraction; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D053838:Scattering, Small Angle
chemical_list: D058619:Maltose-Binding Proteins; D011994:Recombinant Proteins; D017242:Complement Factor H; D003903:Deuterium
keywords: Contrast variation; Matchout deuteration; Neutron scattering; SANS