Thesis Title: Subtitle - NMR Spectroscopy Research Group

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42 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. Figure 2.5 PCS measurements in isopropyl groups of Val and Leu and use of PCS for stereospecific resonance assignments. (a) Selected spectral region from a 2D (H)C(C)H TOCSY spectrum (red) of cz- 186/ loaded with a 1:1 mixture of La 3+ and Yb 3+ , showing the methyl cross- peaks of Val96. The spectrum is overlaid with the 13 C-HSQC spectrum (blue). Intraresidual correlations between the cross-peaks of the 1CH3 and 2CH3 groups are identified by dotted lines. The TOCSY spectrum was recorded with 12 ms mixing time. (b) Same spectral region as in (a) taken from the 2D methyl Cz-EXSY spectrum of the same sample recorded with a mixing time of 480 ms. (c) Selected strips from the 3D methyl CZ-EXSY spectrum of cz- 186/ loaded with a 1:1 mixture of La 3+ and Dy 3+ (right panels) aligned with corresponding spectral regions from the 2D methyl CZ-EXSY spectrum (left panels). The strips display the methyl group correlations of Val10 and Leu113. The arrows point from the chemical shifts of the diamagnetic auto-peaks (dd) to the chemical shifts of the exchange peaks (pd), indicating the 13 C PCS. Horizontal lines identify the
2.4 Results. 43 positions of the dd- and pd-peaks in the 1( 13 C) dimension. The line at 26.35 ppm identifies the 13 C- 13 C NOEs with the CH group of Leu113. (d) Assignment of methyl resonances from the comparison of predicted with experimental PCS. For each Val residue, the distances from the lanthanide in Å are indicated for both methyl carbons in the center of the plot. 13 C PCS and 1 H PCS are displayed as filled and open bars, respectively. The residues are sorted according to the predicted 13 C-PCS and the PCS are plotted in the sequence C 1 /H 1 /C 2 /H 2 . Figure 2.5c compares the measurement of 13 C PCS for two residues from strips of 2D and 3D methyl CZ-EXSY spectra. The two experiments are complementary, showing better frequency resolution in the 2D spectrum and generally less cross-peak overlap in the 3D spectrum. The example of Leu113 shows that one- and two-bond 13 C- 13 C NOE correlations are visible, but generally of much smaller intensity than the exchange peaks. Through-bond correlations can again be identified from TOCSY spectra. From the combined use of the 2D and 3D Cz-EXSY spectra, all 13 C-HSQC cross-peaks observable for any of the methyl groups of the cz- 186/ /Dy 3+ and cz- 186/ /Yb 3+ complexes could readily be correlated with the corresponding 13 C-HSQC cross-peaks of the cz- 186/ /La 3+ complex, yielding the PCS. Compared to the 13 C-HSQC spectrum, the methyl Cz-EXSY spectra yielded the 13 C chemical shifts in the paramagnetic state for a further 47 methyl groups of the cz- 186/ /Dy 3+ complex with rC-Dy distances as short as 10 Å, leaving only 11 methyl groups completely unobservable due to excessive PRE. For the cz- 186/ /Yb 3+ complex, the methyl Cz-EXSY spectra yielded the 13 C chemical shifts for 7 additional methyl groups with rC-Yb distances as short as 6 Å, leaving only 1 methyl group unobservable. Figure 2.5d compares the predicted and measured PCS for Val methyl groups in the cz- 186/ /Dy 3+ complex. Each residue is characterized by up to 4 PCS values, resulting in the straightforward assignment of 10 out of 12 residues. Only Val39 did not yield unambiguous PCS data due to resonance overlap, and Val65 is too close to the Dy 3+ ion. The assignment of Val65 could be made in the 186/ /Yb 3+ complex, where this residue yields large PCS (Supporting Information). The methyl cross-peaks of Leu residues can be assigned in an analogous way (see below). Importantly, this approach automatically yields the stereospecific assignment of Val and Leu methyl peaks, as long as different PCS are observed for the two prochiral methyl groups. Since the methyl carbons in an isopropyl group are separated by 2.5 Å, this is almost always the case
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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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Page 9 and 10: ix 2.3.3 Manual resonance assignmen
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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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Chapter 5 Conclusion and perspectiv
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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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