Thesis Title: Subtitle - NMR Spectroscopy Research Group

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134 Chapter 5. Conclusion and perspectives. Clearly, a larger benchmark containing proteins of different topology and size is necessary to gain a better understanding of the merit of the pseudocontact shift. We are in the process of creating an artificial one, since PCS can easily be predicted. The parameters that would impact the success of calculation, and that we have to explore are: the level of noise within the data PCS- ROSETTA can tolerate, the influence of the position of the paramagnetic center relative to the protein, and the influence of the relative orientations of different Δχ-tensors. 5.2 The use of PCS for chemical shift assignment The work of Chapter 2 provides proof of principle that PCS can be used for automatic chemical shift assignment when a 3D structure is available. Chemical shifts are sensitive to the local environment. Small variations in the surrounding electronic configuration can have a large impact on the chemical shift values. This makes it extremely difficult to predict chemical shifts accurately. In contrast, the PCS of a spin i is much less affected by similar variations, as the PCS only depends on the spherical coordinates of i with respect to the Δχ-tensor frame. While the program Possum presented in Chapter 2 is limited to methyl groups, the approach used to automatically assign the chemical shifts could be applied to any atom type for which PCS can be measured. The simulated annealing protocol used to solve the multi-dimensional assignment problem has proven to be highly efficient at sampling the possible assignment space and find a solution of lower energy than manual assignments. The scoring scheme used to optimize the assignment is also efficient; only few misassignments were present when multiple lanthanide data sets were available. Even more interesting may be to obtain the Δχ-tensor parameters directly from unassigned chemical shifts. The program Echidna (Schmitz et al., 2006) is capable of simultaneously getting the Δχ-tensor parameters and assigning the paramagnetic NMR spectrum, provided that the diamagnetic spectrum is already assigned. This raises the question whether both the diamagnetic and the paramagnetic spectrum can be assigned, while determining the Δχ-tensor at the same time. A software package for this purpose is currently under development. The idea is to handle various kinds of input information (partial assignment for some residues in the diamagnetic or paramagnetic state, measurement of some unassigned pseudocontact shifts, selective isotope labeling of some amino acids) and use this partial information in a simulated annealing protocol to optimize the assignment while determining the Δχ-tensor parameters. The challenge of this approach is to use the right combination of methods: simulated annealing for the assignment, grid
5.3 The use of PCS for protein docking. 135 search for the coordinates of the paramagnetic center, and singular value decomposition for the determination of the remaining Δχ-tensor parameters. 5.3 The use of PCS for protein docking At present, structural biology groups are investing much effort in producing models of proteins by NMR or X-ray crystallography. Currently more than 48000 crystal structures and almost 7000 NMR structures of proteins have been deposited in the protein data bank. In contrast, the number of protein-protein complexes solved by any of those methods remains low (less than 2500). For X-ray crystallography, the difficulty to co-crystallize a complex is much greater than to obtain crystal structures of the individual proteins. For NMR spectroscopy, the larger molecular weight of complexes presents a problem, making it more difficult to obtain and analyze data. Additionally, the most useful information of intermolecular NOEs often involves amino acid side chains the resonances of which are much harder to assign than the backbone resonances of the protein. For a better insight of the molecular basis of life, the challenge is to understand how individual macromolecules come together to fulfill their tasks in DNA replication, gene expression and regulation, etc. Construction of models for protein-protein, protein-DNA and protein-ligand complexes are necessary. An angle to tackle the problem is the use of a docking program that predicts the binding mode of the complex given the structures of the individual components. The major difficulty of this approach is to comprehensively explore the 6-dimensional space that describes the relative orientation and position of two rigid bodies in space. This presents a challenging sampling problem. Shortage of experimental information to support any model generated is another drawback of the docking approach. Therefore, alternative techniques to provide more experimental information would be important. Measurements of residual dipolar couplings are an efficient way to obtain orientational information between two macromolecules: the macromolecules are weakly aligned using an alignment media. Independent determination of the alignment tensor with respect to the two macromolecules gives direct access to the relative orientation of the two rigid bodies. More interestingly, PCS measurements provide, in addition to orientational information, information on the distance between the two bodies. Determination of the Δχ-tensor with respect to the two molecules and superimposition of the two Δχ-tensor frames yields the relative orientation and position of the two macromolecules, as illustrated in Figure 1.8
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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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2 Chapter 1. Introduction. The scal
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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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3.6 Program Features. 81 each fitti
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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