Poster Presentation The 44th Lorne Conference on Protein Structure and Function 2019

The National Deuteration Facility: isotopic labelling of proteins for structure function applications. (#277)

Karyn L Wilde 1 , Anthony P Duff 1 , Natalia Davydova 1 , Agata Rekas 1 , Peter J Holden 1 , Tamim A Darwish 1
  1. National Deuteration Facility - ANSTO, Lucas Heights, NSW, Australia

The National Deuteration Facility (NDF) at the Australian Nuclear Science and Technology Organisation (ANSTO) is the only facility of its type in the Southern Hemisphere, having the specialised expertise and infrastructure for both biological and chemical molecular deuteration. Deuterium (2H or D) is a naturally occurring stable isotope of hydrogen (1H or H) which contains a neutron in addition to the proton and electron found in the naturally abundant protium 1H. Molecular deuteration of molecules significantly increases the options in complex structure function investigations using NMR, neutron scattering, MS and other techniques. Deuteration can provide greater contrast and improved resolution to assist investigations into the relationship between molecular structure and function of molecules of both biological and synthetic origin.

The Biodeuteration group of the NDF 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 protein has also been produced for NMR studies including AILV-13C-methyl labelled protein with a 2H background and protein with a defined set of amino acids selectively 15N and/or 13C labelled. Further method development is continually undertaken to expand NDF capabilities and thus increase the options available to the Australasian research community.

An overview of the NDF facility will be presented along with some examples of recently published research utilising isotopically labelled protein produced by the NDF [2-7].

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 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).
  7. Emily J. Furlong, Hassanul G. Choudhury, Fabian Kurth, Anthony P. Duff, Andrew E. Whitten, and Jennifer L. Martin. Disulfide isomerase activity of the dynamic, trimeric Proteus mirabilis ScsC protein is primed by the tandem immunoglobulin-fold domain of ScsB. J. Biol. Chem. 293, 5793–5805 (2018)