Thesis Title: Subtitle - NMR Spectroscopy Research Group

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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 <strong>NMR</strong> 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
5.1 The use of PCS for structure determination. 131 a and c). However, the C α rmsd against the native structure was found to be reasonably low (2.25 Å for protein G, 1.89 Å for θ, 2.39 Å for ArgN, 5.03 Å for calmodulin). This provides proof of principle that the PCS alone can direct the correct folding of a protein at the fragment assembly level. The conditions of success remain unclear and should be further analyzed. It could be a combination of the complexity or size of the protein, the number and quality of data sets, the relative orientation of each Δχ-tensor, and the location of the paramagnetic center. Figure 5.1 Capacity of the PCS score, as the only energy term, to fold the protein. The lowest PCS energy structure (blue) is superimposed onto the native structure (white). (a) protein G, (b) θ subunit, (c) ArgN, (d) calmodulin. Figure 5.2 The intersection of isosurfaces defines the position and orientation of peptide fragments in the protein structure. (a) Three PCS of the spin (black) measured with three
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Computational study of proteins wit
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iii Schmitz C, Stanton-Cook MJ, Su
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v Acknowledgements I would like to
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vii Keywords paramagnetic nmr, pseu
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ix 2.3.3 Manual resonance assignmen
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xi List of Figures Figure 1.1 NMR e
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xv List of Abbreviations α Subunit
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3.4 Introduction 3.4 Introduction.
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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