Thesis Title: Subtitle - NMR Spectroscopy Research Group

130 Chapter 5. Conclusion and perspectives.
5.1 The use of PCS for structure determination
Structure determination of proteins remains a major challenge of the post genomic era.
Conventional techniques such as X-ray crystallography and NMR spectroscopy are slow. Hence,
the gap between known protein sequences and known protein structures remains large. Alternative
experimental methods are required to speed up the process. Those methods have to present an
attractive compromise between the efforts required to measure the desired data, and the merit that
data can bring in assisting de novo determination of proteins.
In Chapter 4, it has been demonstrated that PCS data are a potential candidate. It has been
shown that combining a molecular fragment approach with the PCS score leads to the correct
folding of proteins smaller than 146 residues. A benchmark of ten data sets has been compiled for
this work, and the PCS-ROSETTA approach showed success in eight out of the ten cases. The first
case where PCS did not lead to the correct folding concerned a protein having only one data set of
PCS. The importance to measure multiple data sets with different lanthanides has already been
shown in Chapter 2 (to increase the quality of the resonance assignment) and Chapter 3 (to increase
the quality of the fitted Δχ-tensor). It was not surprising that PCS-ROSETTA had difficulties when
working with a single data set. The second case where the fold was incorrect was for the largest
protein of the benchmark: the subunit ε, 186 residues. The size of the protein might be a limiting
factor for the current approach. It is well known that for proteins larger than 150 residues, the size
of the conformational space explodes in the molecular fragment replacement protocol of
ROSETTA. The question whether PCS-ROSETTA is facing the same problem is legitimate. Some
elements of responses to that question are presented in the following sections.
5.1.1 Folding of proteins using only pseudocontact shifts
In order to gain a better understanding of the merit of the PCS, structure calculations with
PCS-ROSETTA have been made with all energy terms switched off, except the PCS score. The
fragment assembly within ROSETTA is hence guided purely by the PCS. While turning off the
normal force field of ROSETTA presents no practical interest, the theoretical results presented in
the following explanations remain interesting.
The protocol generated and identified (by the lowest score) attractive folding for four out of
the ten structure calculations (Figure 5.1). The term ―attractive folding‖ has to be defined in that
context. Obviously, without energy terms such as van der Waals terms or hydrogen bonding, the
resulting structures can exhibit steric clashes (Figure 5.1 a, c, d), or poor β-sheet pairing (Figure 5.1