SEMINAR

Magnetising Proteins for NMR and EPR Spectroscopy

Abstract
Paramagnetic probes present outstanding opportunities for structural biology studies by nuclear magnetic resonance (NMR) spectroscopy. In particular, pseudocontact shifts (PCS) from lanthanide labeled proteins provide unique long-range restraints that help to define the structures of proteins, protein-protein complexes, and protein-ligand complexes (1-3). The value of PCSs will be illustrated with a range of applications, including structure determinations of proteins and protein-ligand complexes, and the merits of different paramagnetic tags will be discussed (4), also with regard to applications in EPR spectroscopy (5).

References
[1] G. Otting (2008) Prospects for lanthanides in structural biology by NMR. J. Biomol. NMR 42, 1-9.
[2] H. Yagi et al. (2013) 3D protein fold determination from backbone amide pseudocontact shifts generated by lanthanide tags at multiple sites. Structure 21, 883-890.
[3] W.-N. Chen et al. (2014) The dengue virus NS2B-NS3 protease retains the closed conformation in the complex with BPTI. FEBS Lett. 588, 2206-2211.
[4] D. Shishmarev and G. Otting (2013) How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic tags? A modelling study. J. Biomol. NMR 56, 203-216.
[5] E. H. Abdelkader et al. (2015) Protein conformation by EPR and NMR spectroscopy using lanthanide tagging of genetically encoded amino acids. Angew. Chemie Int. Ed., under review.

BIOGRAPHY

2007-present: Professor, Research School of Chemistry, Australian National University
2002-2007: ARC Federation Fellow at the RSC, ANU
1992-2002: Professor of Molecular Biophysics, Karolinska Institute, Stockholm, Sweden
1987-1992: Assistant and Oberassistent, Institute of Molecular Biology and Biophysics, ETH-Zürich, Switzerland
1987: PhD, Institute of Molecular Biology and Biophysics, ETH-Zürich, Switzerland

Research Fields and Interests:
Structure and structural changes of proteins in solution underpin function and play a critical role in drug development. We are developing methods for structural biology in solution, in particular by NMR and EPR spectroscopy. This includes the use of cell-free protein synthesis for selective isotope labelling and the incorporation of genetically encoded unnatural amino acids as specific sites for chemical tags, for selective detection by NMR spectroscopy, generation of pseudocontact shifts and chemical cross-linking.

Selected Publications:
Abdelkader, E. H., Feintuch, A., Yao, X., Adams, L. A., Aurelio, L., Graham, B., Goldfarb, D., Otting, G. (2015) Protein conformation by EPR and NMR spectroscopy using lanthanide tagging of genetically encoded amino acids. Angew. Chemie Int. Ed., under review.

Chen, W.-N., Kuppan, K. V., Lee, M. D., Jaudzems, K., Huber, T. and Otting, G. (2015) O-tert-butyltyrosine, an NMR tag for high-molecular weight systems and measurements of submicromolar ligand affinities. J. Am. Chem. Soc. 137, 4581-4586.

Loh, C.-T., Graham, B., Abdelkader, E. H., Tuck, K. L. and Otting, G. (2015) Generation of pseudocontact shifts in proteins with lanthanides using small “clickable” nitrilotriacetic acid and iminodiacetic acid tags. Chem. Eur. J. 21, 5084-5092.

Carruthers, T. J., Carr, P. D., Loh, C.-T., Jackson, C. J. and Otting, G. (2014) Fe3+ located in the dinuclear metallo--lactamase IMP-1 by pseudocontact shifts. Angew. Chemie Int. Ed. 53, 14269-14272.

Su, X.-C., Wang, Y., Yagi, H., Shishmarev, D., Mason, C. E., Smith, P. J., Vandevenne, M., Dixon, N. E. and Otting, G. (2014) Bound or free: interaction of the C-terminal domain of E. coli single-stranded DNA binding protein (SSB) with the tetrameric core of SSB. Biochemistry 53, 1925-1934.