Thesis Title: Subtitle - NMR Spectroscopy Research Group

20 Chapter 1. Introduction.
The determination of protein structures is one of the main challenges of the post genomic
era. The knowledge of structures at atomic detail is a prerequisite to understand how
macromolecular complexes assemble and perform their tasks within living organisms. The
established methods of X-ray crystallography and NMR spectroscopy still require significant
human and financial resources to determine the structure of proteins of interest. Efforts are being
focused on high-throughput methods to speed up the process of characterizing a large number of
proteins (Kobe et al., 2008).
De novo structure prediction software packages such as ROSETTA (Simons et al., 1997)
are quite successful for small proteins (< 100 residues). The large size of the conformational space
to explore makes it difficult, however, to tackle larger proteins. To overcome the ―sampling
problem‖, one approach is to include additional experimental restraints that facilitate the three-
dimensional reconstruction of protein structures. Those restraints must be easier to measure than it
would be to obtain crystals of the target protein, or to measure and assign the NOEs required for the
full determination of the structure.
The pseudocontact shift effect is a candidate for this approach. PCSs can be measured
swiftly and accurately as the chemical shift difference between two spectra, once a paramagnetic
probe has been introduced into the protein. The use of lanthanide binding tags makes these
techniques potentially available to any protein. Several lanthanide tags are now available. For a
recent review, see (Su et al., 2009b). While it is not yet routine to attach lanthanide binding tags to
a protein, several options are possible. Attachment by one or two disulfide bonds (Smith et al.,
1975), attachment at one of the termini of the protein (Donaldson et al., 2001), or even use of a
non-covalent tag as demonstrated by (Su et al., 2009a) can be considered. It is expected that
lanthanide attachment techniques will become routine in the future.
Beyond the process of attachment, the second challenge is to have a tag that is not flexible.
The physical model underlying equation (1.1) is accurate if the Δχ-tensor parameters are constant
over time. This hypothesis could be questioned if small movements of the tag occur. Fluctuation of
the tag produces two undesired effects:
(i) It changes the electronic environment in the vicinity of the lanthanide and
consequently, the orientation or magnitude of the Δχ-tensor. As equation (1.1) is
linear with respect to the axial component, the rhombic component, and the three
Euler angles, changes over time of those five parameters will not affect the way
PCSs are predicted. More precisely, n conformations of the Δχ-tensor occurring