Thesis Title: Subtitle - NMR Spectroscopy Research Group

2.5 Discussion. 49
(Sibille et al., 2002), since PCS are far less affected by local mobility as long as the spins are not
very close to the paramagnetic center. The robustness of PCS with regard to structural variations is
particularly beneficial for the assignment of Met CH3 groups that are notoriously difficult to
assign by conventional methods. The potential of PCS for their assignment has been noted
previously (Bose-Basu et al., 2004).
The assignment strategy presented here requires the determination of the tensor, which
can readily be achieved from 15 N- 1 H correlation spectra by the Platypus algorithm (Pintacuda et al.,
2004). Obtaining resonance assignments of methyl groups in this way is attractive because 15 N- 1 H
correlation spectra of backbone amides and 13 C- 1 H correlation spectra of methyl groups can be
recorded even for high-molecular weight systems (Fiaux et al., 2002, Sprangers et al., 2007).
Alternatively, the -tensor parameters can be determined from assigned diamagnetic NMR
resonances and a set of PCS identified by comparison with the paramagnetic NMR spectrum, either
manually or automatically using the Echidna algorithm (Schmitz et al., 2006). Initial sequence-
specific resonance assignments can, if necessary, be achieved by site-directed mutagenesis (Siivari
et al., 1995, Bose-Basu et al., 2004), for example by mutation of Ile to Val (Wu et al., 2007).
Assignments by PCS are not limited to metal-binding proteins as different techniques have
recently become available that achieve site-specific attachment of lanthanide-tags to proteins
devoid of natural metal binding sites (Ma et al., 2000, Dvoretsky et al., 2002, Wöhnert et al., 2003,
Ikegami et al., 2004, Prudêncio et al., 2004, Leonov et al., 2005, Haberz et al., 2006, Rodriguez-
Castañeda et al., 2006, Su et al., 2006). The use of different tags or attachment at different sites
readily generates very different tensors (Rodriguez-Castañeda et al., 2006) that can highlight
inconsistencies between experimental and back-calculated PCS.
If the exchange between paramagnetic and diamagnetic metal ions is too slow to measure
exchange spectra, the program Possum can be used to assign the methyl groups in the diamagnetic
and paramagnetic state. As expected, the robustness of Possum with regard to small differences
between the atomic coordinates of the protein and its actual structure in solution increases with the
amount of additional data available. In this respect, data from two paramagnetic metal ions are
particularly beneficial, but also information about intraresidual methyl-methyl connectivities or
stereospecific identities of methyl groups in Val, Leu, and Ile residues.
The robustness of assignments made by Possum can further be enhanced by the increased
spectral resolution afforded by 3D NMR spectra which would greatly facilitate the identification of
the corresponding NMR resonances in the diamagnetic and paramagnetic state based on the