Thesis Title: Subtitle - NMR Spectroscopy Research Group

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50 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. criterion that all correlated spins are close in space and therefore experience similar PCS. For example, 3D (H)CCH-TOCSY or NOESY- 13 C-HSQC spectra would resolve several cross-peaks for each methyl group, which can simultaneously be compared with the 3D structure of the protein and the predicted PCS to obtain resonance assignments. For methyl groups in the vicinity of the paramagnetic ion, the observation of correlations can be aided by protonless experiments (Bermel et al., 2006). Conceivably, assignments by PCS can also be achieved for perdeuterated proteins of increased molecular weight containing selectively protonated methyl groups (Rosen et al., 1996). The best spectral resolution in the methyl region of the 13 C-HSQC spectrum would be obtained for CD2H groups (Kainosho et al., 2006).Notably, however, the Cz-EXSY experiments described here allowed us to measure all PCS data in the uniformly 13 C/ 15 N-labeled and fully protonated sample, i.e. the improved spectral resolution of selectively labeled samples was not necessary for our system. In conclusion, resonance assignments of the 13 C-HSQC cross-peaks of methyl groups by PCS induced by a site-specifically attached lanthanide ion present a versatile and convenient technique which can open many opportunities for NMR studies of proteins of known three- dimensional structure. It is anticipated that resonance assignments by this technique will be particularly useful in ligand screening applications. 2.6 Acknowledgement The authors thank Don A. Grundel for source codes of the MAP solver and for useful discussions. M.J. thanks the Humboldt Foundation for a Feodor-Lynen Fellowship. Financial support from the Australian Research Council for project grants, a Federation Fellowship for G.O. and the 800 MHz NMR spectrometer at the ANU is gratefully acknowledged. This work was supported by an award under the Merit Allocation Scheme of the National Facility of the Australian Partnership for Advanced Computing. 2.7 Supporting Information Available
2.8 References. 51 Pulse scheme of a (H)C(C)H-TOCSY experiment for correlations between isopropyl methyl groups, 13 C-HSQC spectra of uniformly, fractionally, and selectively isotope labeled cz- 186/ , diagrams comparing experimental and predicted PCS, a table with the chemical shifts of the methyl groups cz- 186 observed in the presence of La 3+ , Yb 3+ , or Dy 3+ , and tables reporting the number of methyl groups assigned by Possum. This material is available free of charge via the Internet at http://pubs.acs.org. 2.8 References Allegrozzi M, Bertini I, Janik MBL, Lee YM, Lin GH and Luchinat C (2000) Lanthanide-induced pseudocontact shifts for solution structure refinements of macromolecules in shells up to 40 Å from the metal ion. J Am Chem Soc 122:4154-4161 Balas E and Saltzman MJ (1991) An algorithm for the 3-index assignment problem. Oper Res 39:150-161 Balayssac S, Jiménez B and Piccioli M (2006) Assignment strategy for fast relaxing signals: complete aminoacid identification in thulium substituted Calbindin D9K. J Biomol NMR 34:63-73 Bax A, Delaglio F, Grzesiek S and Vuister GW (1994) Resonance assignment of methionine methyl groups and χ 3 angular information from long-range proton-carbon and carbon- carbon J correlation in a calmodulin-peptide complex. J Biomol NMR 4:787-797 Bax A, Ikura M, Kay LE and Zhu G (1991) Removal of F1 baseline distortion and optimization of folding in multidimensional NMR spectra. J Magn Reson 91:174-178 Bermel W, Bertini I, Felli IC, Piccioli M and Pierattelli R (2006) 13 C-detected protonless NMR spectroscopy of proteins in solution. Prog NMR Spectrosc 48:25-45 Bose-Basu B, DeRose EF, Kirby TW, Mueller GA, Beard WA, Wilson SH and London RE (2004) Dynamic characterization of a DNA repair enzyme: NMR studies of [methyl- 13 C]methionine-labeled DNA polymerase β. Biochemistry 43:8911-8922 DeRose EF, Darden T, Harvey S, Gabel S, Perrino FW, Schaaper RM and London RE (2003) Elucidation of the ε- θ subunit interface of Escherichia coli DNA polymerase III by NMR spectroscopy. Biochemistry 42:3635-3644 Dvoretsky A, Gaponenko V and Rosevear PR (2002) Derivation of structural restraints using a thiol-reactive chelator. FEBS Lett 528:189-192
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Computational study of proteins wit
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iii Schmitz C, Stanton-Cook MJ, Su
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v Acknowledgements I would like to
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vii Keywords paramagnetic nmr, pseu
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ix 2.3.3 Manual resonance assignmen
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xi List of Figures Figure 1.1 NMR e
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