Thesis Title: Subtitle - NMR Spectroscopy Research Group

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48 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. correct assignment could have been obtained for Leu95 in the Yb 3+ complex (Figure 2.6b). None of the other Val and Leu residues swapped their stereospecific assignment when we changed their 1 angles ( 2 angles in the case of Leu) by ±10º. In the case of Leu11, different NMR criteria suggest that its side chain undergoes dynamic conformational averaging. (i) The 13 C-PCS values predicted from the structure are -7.0 and -11.9 ppm, whereas the experimental value found for both methyl groups is -8.4 ppm. (ii) In the crystal structure, the two methyl groups are 9.2 and 11.1 Å from the metal ion, but the 13 C-NMR line widths observed for the methyl groups in the cz- 186/ /Dy 3+ complex are indistinguishable. (iii) Both methyl resonances overlap with each other in the 13 C-HSQC spectrum, indicating similar chemical environments, and their line shapes are narrower than those of most other methyl groups. Remarkably, however, Leu11 is located in the hydrophobic core of the protein and is very well defined in the crystal structure, 40 although the side chain forms no steric contacts and could access different rotameric states without introducing van der Waals violations with neighboring atoms. Conceivably, the low temperature used in the X-ray experiment may have frozen out a single conformation, whereas a much larger conformational space is accessible at room temperature. Ile154 presents an example where partial motional averaging may be indicated by a smaller difference observed between the PCS of the 1 and 2 carbon atoms than predicted. The side chain heavy atoms of this residue shows enhanced B-factors in the crystal structure, in agreement with its location at the protein surface. 2.5 Discussion The present work shows that methyl resonances of 13 C-labeled proteins can be assigned solely from PCS with reference to the 3D structure of the protein, yielding both sequence- and stereo-specific resonance assignments without having to establish connectivities to backbone resonances. This presents a significant advance over our previous strategy for the assignment of 15 N-HSQC spectra, which relied on PCS, PRE, CCR, and RDCs measured on selectively labeled samples (Pintacuda et al., 2004). Clearly, any resonance assignment based on comparison of experimental and back- calculated PCS critically depends on the accuracy of the 3D structure of the protein and is expected to fail for flexible protein segments. Yet, this problem is much less severe than in the case of RDCs
2.5 Discussion. 49 (Sibille et al., 2002), since PCS are far less affected by local mobility as long as the spins are not very close to the paramagnetic center. The robustness of PCS with regard to structural variations is particularly beneficial for the assignment of Met CH3 groups that are notoriously difficult to assign by conventional methods. The potential of PCS for their assignment has been noted previously (Bose-Basu et al., 2004). The assignment strategy presented here requires the determination of the tensor, which can readily be achieved from 15 N- 1 H correlation spectra by the Platypus algorithm (Pintacuda et al., 2004). Obtaining resonance assignments of methyl groups in this way is attractive because 15 N- 1 H correlation spectra of backbone amides and 13 C- 1 H correlation spectra of methyl groups can be recorded even for high-molecular weight systems (Fiaux et al., 2002, Sprangers et al., 2007). Alternatively, the -tensor parameters can be determined from assigned diamagnetic NMR resonances and a set of PCS identified by comparison with the paramagnetic NMR spectrum, either manually or automatically using the Echidna algorithm (Schmitz et al., 2006). Initial sequence- specific resonance assignments can, if necessary, be achieved by site-directed mutagenesis (Siivari et al., 1995, Bose-Basu et al., 2004), for example by mutation of Ile to Val (Wu et al., 2007). Assignments by PCS are not limited to metal-binding proteins as different techniques have recently become available that achieve site-specific attachment of lanthanide-tags to proteins devoid of natural metal binding sites (Ma et al., 2000, Dvoretsky et al., 2002, Wöhnert et al., 2003, Ikegami et al., 2004, Prudêncio et al., 2004, Leonov et al., 2005, Haberz et al., 2006, Rodriguez- Castañeda et al., 2006, Su et al., 2006). The use of different tags or attachment at different sites readily generates very different tensors (Rodriguez-Castañeda et al., 2006) that can highlight inconsistencies between experimental and back-calculated PCS. If the exchange between paramagnetic and diamagnetic metal ions is too slow to measure exchange spectra, the program Possum can be used to assign the methyl groups in the diamagnetic and paramagnetic state. As expected, the robustness of Possum with regard to small differences between the atomic coordinates of the protein and its actual structure in solution increases with the amount of additional data available. In this respect, data from two paramagnetic metal ions are particularly beneficial, but also information about intraresidual methyl-methyl connectivities or stereospecific identities of methyl groups in Val, Leu, and Ile residues. The robustness of assignments made by Possum can further be enhanced by the increased spectral resolution afforded by 3D NMR spectra which would greatly facilitate the identification of the corresponding NMR resonances in the diamagnetic and paramagnetic state based on the
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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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Page 15: xv List of Abbreviations α Subunit
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Page 42 and 43: 26 Chapter 1. Introduction. Su XC,
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Page 95 and 96: 3.5 Algorithm. 79 rhombic component
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Page 99 and 100: 3.6 Program Features. 83 A new PCS
Page 101 and 102: 3.6 Program Features. 85 Carlo prot
Page 103 and 104: 3.7 Study case. 87 The coordinates
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Page 109 and 110: 3.9 Acknowledgment. 93 variables, t
Page 111 and 112: 3.10 References. 95 Galassi M, Davi
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3.11 Supporting information. 99 Tab
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102 Chapter 4. Protein Structure De
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Chapter 5 Conclusion and perspectiv
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5.1 The use of PCS for structure de
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5.1 The use of PCS for structure de
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5.3 The use of PCS for protein dock
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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