Thesis Title: Subtitle - NMR Spectroscopy Research Group

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38 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. 2.4.2 Methyl CZ-EXSY experiments In order to measure PCS data conveniently and with high sensitivity, we designed an experiment applicable to samples prepared with a 1:1 mixture of paramagnetic and diamagnetic metal ions, where chemical exchange between the metal ions leads to exchange of the protein between paramagnetic and diamagnetic states. By generating exchange cross-peaks between methyl peaks of the diamagnetic and paramagnetic lanthanide complexes, the experiment allows the measurement of 1 H and 13 C PCS from a single spectrum. Figure 2.1 shows 2D and 3D versions of the methyl Cz-EXSY experiment. The pulse sequences are related to previously published Nz- exchange experiments (Farrow et al., 1994, John et al., 2007a). During a mixing period m, magnetization is stored as relatively slowly relaxing CZ magnetization. Simulations indicate that, owing to rapid rotation around the 3-fold symmetry axis, longitudinal relaxation rates R1 of methyl 13 C spins are fairly insensitive with respect to molecular size, and barely exceed 2 s –1 even for very small proteins (Figure 2.1d). In the cz- 186/ /La 3+ complex ( C = 17 ns), we measured R1( 13 C) rates of about 1.6 s –1 for the majority of methyl groups. Only a group of highly mobile Thr residues relaxed somewhat faster (2 s –1 ), whereas the R1( 13 C) relaxation in Met CH3 groups was much slower (about 0.7 s –1 ). In contrast to transverse relaxation rates R2, R1 rates in macromolecules are barely affected by the paramagnetism of lanthanides(John et al., 2007a). The experiment yields auto-peaks for the diamagnetic and paramagnetic states (dd and pp peaks, respectively) and exchange peaks arising from magnetization exchange from the paramagnetic to the diamagnetic state and vice versa (pd and dp peaks, respectively). Since the experiment starts from 13 C polarization rather than using an INEPT transfer, pd peaks can be detected even for methyl groups that are strongly affected by 1 H PRE in the paramagnetic state and thus invisible in the 13 C-HSQC spectrum. Combined with the dd peaks, this allows 13 C PCS measurements that are limited only by the (16-fold smaller) 13 C PRE (John et al., 2007b). As indicated previously 13 and illustrated by the simulations of Figure 2.1e, 13 C polarization in methyl groups of proteins can be very efficiently enhanced using the { 1 H} 13 C NOE. This holds irrespective of paramagnetism. We observed an about two-fold increase in 13 C polarization in the cz- 186/ /La 3+ complex using 1 s of 1 H irradiation between subsequent scans. For improved resolution in the 13 C dimension and measurement of small 13 C PCS, the 2D experiment is implemented as a constant-time experiment in the t1 dimension. The 3D experiment additionally records the 13 C frequency of the protein state after the mixing time. Real-time
2.4 Results. 39 evolution periods in both indirect dimensions yield superior sensitivity for residues with substantial 13 C PRE that commonly also have larger PCS. Selective 13 C pulses select the spectral window of the methyl 13 C resonances of the diamagnetic complex in order to limit the spectral width required in the F1 dimension. 2.4.3 Resonance assignment of Met, Ala and Thr methyl groups Figure 2.4a shows the spectral region of the Met CH3 cross-peaks of the 2D methyl Cz- exchange spectrum, recorded with a sample of cz- 186/ containing La 3+ and Dy 3+ in a 1:1 ratio. Out of six Met residues, four are observed in the 13 C-HSQC spectrum of the cz- 186/ /Dy 3+ complex (the cross-peak of Met178 in the paramagnetic state appears with very weak intensity at 2( 1 H) = 5.33 ppm). For these residues, both auto and both exchange peaks become visible in the exchange spectrum, forming a rectangle that allows straightforward identification of dd-pp peak pairs, yielding 13 C PCS of 3.28, 1.31, 0.49 and –0.65 ppm. A fifth residue only yields a pd exchange peak with a 13 C PCS value of –3.39 ppm. Figure 2.4 Assignment of Met CH3 from PCS. (a) 2D methyl Cz-EXSY spectrum of cz- 186/ (containing 13 C/ 15 N-labeled cz- 186) loaded with a 1:1 mixture of La 3+ and Dy 3+ (red), overlaid with the 13 C-HSQC spectrum (blue). The diamagnetic auto-peaks (dd) are labeled with the assignment and connected to the paramagnetic auto-peaks (pp) and the exchange peaks (pd and dp) with dashed rectangles. The dp and pp peaks of Met178 are outside the selected spectral region
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Computational study of proteins wit
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Page 9 and 10: ix 2.3.3 Manual resonance assignmen
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3.10 References. 95 Galassi M, Davi
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Thesis Title: Subtitle - NMR Spectroscopy Research Group

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