Thesis Title: Subtitle - NMR Spectroscopy Research Group

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40 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. at 2 = 5.53 ppm. Met107 only shows a pd exchange peak (vertical dashed line), and neither pp- peak nor exchange peaks are observed for Met18. The spectrum was recorded with a mixing time of 480 ms. (b) Comparison of predicted (top) and measured (bottom) PCS of Met CH3 groups. 13 C PCS and 1 H PCS are plotted with filled and open bars, respectively, and sorted according to the predicted 13 C PCS. The distances rC-Ln are given in Å in the center. 3 The measured PCS can be compared with values predicted from the known structure of 186 (Park, 2006) and the previously determined tensor of Dy 3+ (Figure 2.4b) (Schmitz et al., 2006). Only five Met residues belong to the structured part of the protein with predicted 13 C PCS of 3.74 (Met178), 1.30 (Met137), –0.56 (Met87), –1.68 (Met18) and –2.87 ppm (Met107). Met185 is located in the flexible cyclizing loop of cz- 186 and can be immediately assigned to the very intense and narrow resonance with a 13 C PCS of 0.49 ppm, in agreement with the PCS of 0.53 ppm observed for the amide proton of this residue. Met18 is the residue closest to the metal ion (rC-Dy = 12.0 Å) and can be assigned to the methyl group that does not show any exchange peak. As Met18 lines the active site this assignment is independently confirmed by its sensitivity to titration with nucleotides (unpublished results). Met107 is the second closest residue (rC-Dy = 14.2 Å) and displays a pd but no dp exchange peak; the assignment of all other Met residues follows in a straightforward manner from the PCS data. The data show that it is possible to assign a limited number of methyl groups using PCS only. The situation is more complex for the methyl groups of the other amino acids since with the exception of Ile CH3 groups, the amino acid type cannot be identified from 13 C-HSQC spectra alone. This information would have to be provided either by the use of residue-specific labeling or additional <strong>NMR</strong> experiments (in the cz- 186/ /La 3+ complex, the amino acid type can readily be identified from a 3D (H)CCH-TOCSY spectrum). In addition, important information is provided by (i) the relative size of 13 C and 1 H PCS and (ii) whether the paramagnetic 1 H resonance can be observed (rC-Dy > 15 Å) or only pd exchange peaks (rC-Dy > 10 Å, Figure S2.4 and Figure S2.5). 3 Experimental PCS have an error below 0.1 ppm. Errors in the calculated PCS depend on the quality of the 3D structures used. Residues 87, 107, 137 and 178 belong to structured part. The error on their calculated PCS can be considered below 10% of their absolute value.
2.4.4 Assignments of Val, Leu, and Ile methyl groups 2.4 Results. 41 Val, Leu, and Ile are amino acids with two methyl groups that can easily be linked by correlations observed in TOCSY spectra; combining the PCS data for both methyl groups greatly facilitates the resonance assignment of these residues. This is illustrated in Figure 2.5 with the cz- 186/ complex containing 1:1 mixtures of Yb 3+ /La 3+ (a, b) and Dy 3+ /La 3+ (c, d), respectively. Whereas a 2D (H)C(C)H-TOCSY experiment (Figure S2.1, Supporting Information) recorded with short mixing time (12 ms) strongly favors intra-residual methyl-methyl correlations (Figure 2.5a) (Eaton et al., 1990), the 2D methyl CZ-EXSY spectrum yields predominantly exchange peaks and only weak 1- 2 correlations arising from 13 C- 13 C NOE (Fischer et al., 1996). We have also applied the (H)C(C)H-TOCSY experiment to a sample of the pure cz- 186/ /La 3+ complex containing selectively 13 C/ 15 N-Leu labeled cz- 186, where all 1- 2 methyl pairs could be identified (Figure S2.4).
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Computational study of proteins wit
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iii Schmitz C, Stanton-Cook MJ, Su
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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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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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