Thesis Title: Subtitle - NMR Spectroscopy Research Group

132 Chapter 5. Conclusion and perspectives.
different lanthanides can be depicted as three isosurfaces (red, blue and yellow) where the
spin must be located. In order to fulfill all three PCS data simultaneously, the spin must be
located at the intersection of the three isosurfaces, helping to define the orientation and
position of the fragment (purple) in the protein structure (white). (b) The same principle
holds, if PCS from different lanthanide binding sites are available, in which case the
intersection of the three isosurfaces is even better defined.
A direct consequence of those results is the theoretical demonstration that it is possible to
apply the PCS score in a ―divide and conquer‖ approach in order to overcome the sampling
problem typically encountered with proteins larger than 120 residues. If a large protein was
composed of the four proteins present in Figure 5.1, a protocol to obtain the folding of the large
protein could be (i) to cut the protein in four pieces (Figure 5.1 a-b-c-d), (ii) to reassemble the
pieces separately, and (iii) to reconstitute the whole proteins by superimposition of the four
separately determined (sets of) Δχ-tensors. The proof of principle holds as no energy term (that
would report on interactions between the four parts of the proteins) is used. A more efficient way
however, could be to work with smaller overlapping fragments. The size of the fragments would
need to be large enough to make it possible to optimize the Δχ-tensor (larger than 20 residues), but
not too small to prevent running into the sampling problem (smaller than 80 residues).
Clearly, the PCS-score would benefit favorably from additional energy terms, starting from
the van der Waals term that would strongly penalize conformations with steric clashes, thus
favoring the sampling of the correct fold. In particular, the symmetric shape of the isosurfaces
implies the existence of symmetrical folds that could be discriminated with the help of the van der
Waals term, as theoretically demonstrated in (Bertini et al., 2002).
5.1.2 Uses of multiple lanthanide binding sites
Erstwhile limited to metalloproteins, paramagnetic NMR is increasingly enjoying a wider
playground since the arrival of lanthanide binding tags. These tags can be attached to the protein
via a disulfide bond or at the termini of the protein. A non-covalent tag can also be used, although
the disadvantage is the loss of control over whether (and where) the tags bind. Attaching metal tags
to a protein of interest site-specifically is one of the current challenges of paramagnetic NMR.
Several groups are developing new tags, for a recent review, see (Su et al., 2009). While those tags
are engineered to simplify as much as possible the process of tag attachment, a consequence of this