Thesis Title: Subtitle - NMR Spectroscopy Research Group

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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 <strong>NMR</strong> 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 <strong>NMR</strong>. 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
5.1 The use of PCS for structure determination. 133 quest will hopefully be the possibility to attach them at different positions on the surface of the target protein. All work outlined in this thesis has greatly benefited from the availability of multiple data sets measured with different lanthanides. The magnitude of the paramagnetic dipole moment differs between different paramagnetic metal ions. The orientation of the Δχ-tensor varies too. The advantage is that those differences provide additional information that can be used to improve the quality of the fitted Δχ-tensor (Chapter 3) or the quality of the automated assignment (Chapter 2). PCS-ROSETTA calculations can take advantage of that fact. Especially the calculations done on calbindin greatly improved when all available lanthanide data were used compared to test calculations using PCS from only one or two lanthanides simultaneously. It can be expected that the use of tags attached at different locations on the protein will enhance the PCS-ROSETTA calculations further. In particularly, the location and the orientation of fragments with respect to the rest of the protein structure would be defined more accurately by isosurfaces intersecting at steeper angles (Figure 5.2). The current implementation of PCS-ROSETTA would make it straightforward to design such a protocol. The benefits of using two different lanthanide binding sites could be explored already using the arginine repressor as a test case, were PCS data measured for two different lanthanide binding sites are available. 5.1.3 Development of a new PCS-ROSETTA protocol The way structures are calculated by PCS-ROSETTA is similar to the standard protocol of ROSETTA. The only difference is the calculation of a PCS-score during the fragment assembly stage (which requires fitting of a Δχ-tensor and metal position). The weight of the PCS-score compared relative to the standard centroid score of ROSETTA is chosen so that both have an equal influence. While ROSETTA benefits from additional experimental restraints such as PCSs, it is important to bear in mind that the original goal of ROSETTA is to generate a wide variety of protein-like structures in a first step (fragment assembly) and identify the native one in a second step (full atom score). Considering that the PCS have proven to drive the sampling towards the native structure with great efficiency, it can be questioned whether it is necessary to enforce diversity in the generated structures. Several thousands of structures (using a large amount of CPU time) are usually generated by ROSETTA to cover a wide range of possible structures. It may be more profitable to generate, at equal CPU time, a smaller number of structures for which more time is spent for the fragment assembly.
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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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Chapter 3 Numbat: new user-friendly
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3.4 Introduction 3.4 Introduction.
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3.5 Algorithm. 79 rhombic component
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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