Protein labelling (<sup>2</sup>H/<sup>13</sup>C/<sup>15</sup>N) at the National Deuteration Facility supporting structural investigations using NMR. — ASN Events
  1. Schedule
  2. Session 7: Poster Session B - Including Happy Hour & Trade Display
  3. Protein labelling (2H/13C/15N) at the National Deuteration Facility supporting structural investigations using NMR.

Protein labelling (2H/13C/15N) at the National Deuteration Facility supporting structural investigations using NMR. (#234)

Karyn L Wilde 1 , Vanessa K Morris 2 , Rasmus Linser 3 , Ann H Kwan 4 , Margaret Sunde 5 , Lu Li 6 , Emma J Petrie 6 , Paul R Gooley 6 , Anthony P Duff 1 , Peter J Holden 1
  1. National Deuteration Facility, ANSTO, Sydney, Australia
  2. Department Chemie Festkörper-NMR-Spektroskopie, Technische Universität München, Garching, Germany
  3. School of Chemistry, University of New South Wales, Sydney, Australia
  4. School of Molecular Biosciences, The University of Sydney, Sydney, Australia
  5. School of Medical Sciences, The University of Sydney, Sydney, Australia
  6. Department of Biochemistry and Molecular Biology, The University of Melbourne, Melbourne, Australia

The National Deuteration Facility (NDF*) has developed expertise in biological molecular deuteration for the in vivo deuteration of proteins, where all or a fraction of the hydrogen atoms in an expressed protein are replaced by the stable isotope deuterium (2H).


Of particular interest to the NMR community for both solution and solid-state NMR applications, is the NDF capability of the production of multiply-labelled proteins using 13C and 15N. As with deuterium, the isotopes 13C and/or 15N are introduced into the minimal growth medium utilised for biomass production and protein expression using the NDF methods for high-yield recombinant expression of partial or per-deuterated protein in Escherichia coli.


Along with an overview of the NDF labelling methods, examples of multiple labelled expression will be highlighted including the triple-labelled fungal protein EASdelta15 and the double-labelled low density lipoprotein, LDLa. EASdelta15 is a hydrophobin that self-assembles into monolayers at surfaces and interfaces. These monolayers are composed of laterally-assembled fibrils that are a form of functional amyloid. EASdelta15 was triple-labelled (2H/13C/15N) for solid state NMR analysis1, in order to investigate the molecular structure of the fibrillar form. LDLa is a protein module of the G-protein coupled receptor (GPCR) RXFP1which is essential for activation when RXFP1 binds to the hormone relaxin (which is in clinical trials for the treatment of heart failure). LDLa was double-labelled (2H /15N) for Saturation Transfer Difference (STD) NMR experiments to investigate the surface of the LDLa module that interacts with the receptor.

*The NDF offers facilities, staff and expertise to the neutron beam and NMR user communities through externally refereed proposal schemes, accessible at http://www.ansto.gov.au/ndf

1 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 β–sheet core amidst structural heterogeneity. Angewandte Chemie Int Ed  2012, 51: 12621-12625