Thesis Title: Subtitle - NMR Spectroscopy Research Group

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