Thesis Title: Subtitle - NMR Spectroscopy Research Group

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36 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. para δ S exp), Possum will compute the two possible costs and only keep the lower one. (d) For Ile, Leu, and Val residues, the methyl-methyl connectivity information may be available in the diamagnetic state but not in the paramagnetic state. This situation creates a four-dimensional assignment problem for data from a single paramagnetic lanthanide. The program also takes into account the absence of paramagnetic peaks due to PRE by preventing the assignment of observable paramagnetic peaks to methyl groups located closer to the metal ion than a user-specified cutoff. In the present work, cutoffs of 6 and 9 Å were used for the Yb 3+ and Dy 3+ complexes, respectively. Paramagnetic peaks missing for any other reason (e.g. spectral overlap) are also tolerated. This is achieved by assigning a cost only to pairings of observable paramagnetic and diamagnetic peaks, whereas a zero cost is associated with any unassigned diamagnetic peak left over. Finally, the program allows for the possibility that paramagnetic shifts may have been observed only for either the 13 C or the 1 H resonance of a methyl group. The calculation of the assignment starting from the chemical shifts of Table S2.1 took less than 2 h on an AMD 64 4200+ processor, when using all available information, including methyl connectivity and methyl specificity information and the chemical shifts from the Yb 3+ and Dy 3+ complexes. 2.4 Results 2.4.1 13 C-HSQC spectra of the cz- 186/ /Ln 3+ complexes Constant-time 13 C-HSQC spectra of the uniformly 13 C labeled diamagnetic cz- 186/ /La 3+ complex and the paramagnetic cz- 186/ /Dy 3+ and cz- 186/ /Yb 3+ complexes illustrate the spectral complexity of the methyl region and the effect of the paramagnetism. The spectrum of the cz- 186/ /La 3+ complex (blue peaks in Figure 2.3) contains approximately the number of methyl peaks expected for 19 Ala, 14 Thr, 6 Met, 12 Val, 17 Leu, and 14 Ile residues (125 methyl groups). The signals of Met CH3 groups are particularly well resolved and easily identified as they appear with opposite sign.
2.4 Results. 37 Figure 2.3 Methyl region of constant-time 13 C-HSQC spectra of the cz- 186/ complex (containing 13 C/ 15 N labeled cz- 186) in the presence of La 3+ (blue) and a 1:1 mixture of (a) La 3+ /Dy 3+ and (b) La 3+ /Yb 3+ (red). Met CH3 and CH2 groups appear with inverted sign (light colors). The spectra were recorded using a constant time of 28 ms and t2max = 160 ms. The spectra of the mixed samples were acquired with 4 times as many scans to compensate for the halving of the effective concentrations. As Dy 3+ is one of the strongest paramagnetic lanthanide ions (Pintacuda et al., 2007), the methyl peaks of the cz- 186/ /Dy 3+ complex (red peaks in Figure 2.3a) are strongly shifted by PCS and affected by 1 H line broadening due to transverse paramagnetic relaxation enhancement (PRE). Thus, only 55 cross-peaks are observable corresponding to methyl groups with a distance from the Dy 3+ ion larger than 15 Å, many of them with intensities close to the noise level. Part of the 1 H line broadening is caused by unresolved RDCs, including intra-methyl RDCs (Kaikkonen et al., 2001), originating from the paramagnetically induced alignment of the protein with the magnetic field. In the cz- 186/ /Yb 3+ complex, the cutoff distance is reduced to about 9 Å due to the about 6 times smaller paramagnetic moment of Yb 3+ so that only 10 methyl peaks are expected to be broadened beyond detection. Of the remaining 115 methyl resonances (red peaks in Figure 2.3b), only 14 peaks could not be analyzed due to overlap or very small PCS at larger distances from the metal ion. Figure 2.3 shows that for both paramagnetic lanthanides, it is nearly impossible to trace the paramagnetic shift of a 13 C-HSQC peak using the criterion that the PCS in the 13 C and 1 H dimensions of the spectrum must be similar. (In methyl groups, the distance between the carbon and the average position of the three protons is less than 0.4 Å.) Therefore, without prior knowledge of resonance assignments, PCS measurements cannot be made manually from 13 C- HSQC spectra alone.
Page 1 and 2: Computational study of proteins wit
Page 3 and 4: iii Schmitz C, Stanton-Cook MJ, Su
Page 5 and 6: v Acknowledgements I would like to
Page 7 and 8: vii Keywords paramagnetic nmr, pseu
Page 9 and 10: ix 2.3.3 Manual resonance assignmen
Page 11 and 12: 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 95 and 96: 3.5 Algorithm. 79 rhombic component
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3.7 Study case. 87 The coordinates
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3.7 Study case. 89 illustrates how
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3.7 Study case. 91 can be used to a
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3.9 Acknowledgment. 93 variables, t
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3.10 References. 95 Galassi M, Davi
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3.11 Supporting information. 97 Zwe
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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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5.3 The use of PCS for protein dock
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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