Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Protein labelling strategies at The National Deuteration Facility: isotopic labelling for NMR (2H/13C/15N) and neutron (2H) structural investigations (#284)

Karyn L Wilde 1 , Anthony P Duff 1 , Natalia Davydova 1 , Agata Rekas 1 , Tamim Darwish 1 , Peter J Holden 1
  1. National Deuteration Facility, ANSTO, Sydney, Australia

The National Deuteration Facility (NDF) at the Australian Nuclear Science and Technology Organisation (ANSTO) has developed reliable and robust methods for the deuteration and multiple isotope labelling of a broad range of proteins by recombinant expression in Escherichia coli BL21, for the support of neutron scattering and solution or solid-state NMR studies of proteins [1].  Deuterium (2H) and the isotopes 13C and/or 15N are introduced into our minimal defined growth medium utilised for biomass production using NDF methods for high-yield recombinant protein expression [1].  Partially deuterated and perdeuterated protein is produced for small angle neutron scattering (SANS) and neutron crystallography investigations, with triple- (2H/15N/13C) and double-labelled (2H/15N) protein produced for NMR studies.  Selectively-labelled (15N/13C) protein has also been produced for NMR studies, with further method development continually undertaken to expand NDF capabilities. For example, production of ILV- and AILV-13C-methyl labelled protein with a 2H background is currently being developed and demonstrated with collaborator proposals.

An overview of the NDF facility and labelling methods will be presented along with some brief examples of published research utilising deuterated and multiple-labelled protein produced by the NDF [2-6].

Access to the NDF is available through an externally refereed proposal scheme.  For further detail and information refer to http://www.ansto.gov.au/ndf . 

The National Deuteration Facility is partially funded by the National Collaborative Research Infrastructure Strategy (NCRIS) – an initiative of the Australian Federal Government.

  1. Duff AP, Wilde KL, Rekas A, Lake V, Holden PJ. Robust high-yield methodologies for 2H and 2H/15N/13C labelling of proteins for structural investigations using neutron scattering and NMR. Methods in Enzymology, 565, 3-25 (2015).
  2. Morris VK, Linser R, Wilde KL, Duff AP, Sunde M, Kwan AH. Solid-state NMR spectroscopy of functional amyloid from a fungal hydrophobin: a well-ordered –sheet core amidst structural heterogeneity. Angewandte Chemie Int Ed, 51, 12621-12625 (2012).
  3. Christie MP, Whitten AE, King GJ, Hu SH, Jarrott RJ, Chen KE, Duff AP, Callow P, Collins BM, James DE, Martin JL. Low-resolution solution structures of Munc18:Syntaxin protein complexea indicate an open binding mode driven by the Syntaxin N-peptide. PNAS, 109, 9816-9821 (2012)
  4. Taylor JE, Chow JYH, Jeffries CM, Kwan AH, Duff AP, Hamilton W, Trewhella J. Calmodulin binds a highly extended HIV-1 MA protein that refolds upon its release. Biophys. J, 103, 541-549 (2012).
  5. Golden E, Attwood PV, Duff AP, Meilleur F, Vrielink A. Production and characterization of recombinant perdeuterated cholesterol oxidase. Anal Biochem, 485, 102-108 (2015).
  6. Chen X, Wilde KL, Wang H, Lake V, Holden PJ, Middelberg APJ, He L, Duff AP. High yield expression and efficient purification of deuterated human protein galectin-2. Food and Bioproducts Processing, 90, 563-572 (2012).