Poster Presentation Australian & New Zealand Society of Magnetic Resonance Conference 2017

Analysis of isotope incorporation and automated methyl assignment using a f-2H-[U-13C,15N]-labelled sample produced by Australia’s National Deuterium Facility (#113)

Biswaranjan Mohanty 1 , Stefan Nebl 1 , Bradlley C. Doak 1 , Gaurav Sharma 1 , Martin L. Williams 1 , Martin J. Scanlon 1
  1. Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia

The current study reports NMR data generated for a 22 kDa protein from Burkholderia pseudomallei (BpsDsbA), which was expressed in D2O media using a protocol developed by Australia’s National Deuteration Facility (NDF).1 Nearly 40 g wet cell pellet was obtained per 1 L bioreactor culture using non-deuterated 13C-glycerol and 15NH4Cl as carbon and nitrogen source respectively, yielding ~ 60 mg purified protein/10 g cell pellet. Firstly, we characterised the isotope incorporation at the individual Hα, Hβ and methyl sites of the protein sample generated using the NDF protocol in comparison with the protein expressed using non-deuterated 13C-glucose in D2O media, as described previously.2 We have previously described a strategy to assign the ATILV methyl groups of the protein from a single fractional deuterated NMR sample.3 We have now developed an algorithm to automatically generate the side-chain methyl assignments for all methyls except Metε, which requires a 3D NUS CHD2-C-TOCSY peak list and Cα and Cβ assignments obtained previously. For BpsDsbA, we obtained >85% agreement between automated and manual assignments. The automated assignments were generated within a few minutes from the known Cα and Cβ assignments and the C-TOCSY peak list. We found that missing assignments were mainly due to the incomplete Cα and Cβ resonances and/or residues which had very similar 13Cαβ shifts (mostly observed for alanine and leucine amino acid residues). Since the Hβ of alanine amino acid residues are ~50% protonated, in this case an additional 3D NUS-HBHA(CO)NH spectrum was acquired to resolve the ambiguous Ala-CHD2 assignments. In addition, we have implemented a new 3D NUS CHD2-[1H,1H]-NOESY-ct-HSQC pulse sequence to resolve the ambiguous and missing CHD2-methyl assignments using the available crystal structure of the target protein. These methyl assignments have been used to determine the location of ligand binding.

  1. Duff et al. Robust high-yield methodologies for 2H and 2H/15N/13C labelling of proteins for structural investigations using neutron scattering and NMR; Methods Enzymol., 565, 3–25, 2015.
  2. Otten et al. Comprehensive and cost-effective NMR spectroscopy of methyl groups in large proteins; J. Am. Chem. Soc. 132, 2952–2960, 2010.
  3. Mohanty et al. An efficient method determining the binding mode of small molecules in weak protein–ligand complexes; J. Biomol. NMR. 66, 195–208, 2016