Thesis Title: Subtitle - NMR Spectroscopy Research Group

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30 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. coli DNA polymerase III. The active site of 186 binds two divalent ions (Hamdan et al., 2002b) that can be replaced by a single Ln 3+ ion (Pintacuda et al., 2004). 2.3 Experimental section 2.3.1 Sample preparation A cyclized version of 186, cz- 186, was designed for enhanced stability of the protein in unrelated crystallographic experiments (Park, 2006). Using an intein-based strategy (Williams et al., 2002), the N-terminal Ser2 and C-terminal Ala186 of 186 were linked by the nonapeptide TRESGSIEF (numbered 187-195). Apart from the N- and C-terminal residues that are structurally disordered in 186 (Hamdan et al., 2002b),the amide proton chemical shifts of the linear protein are conserved in cz- 186 within ±0.05 ppm, indicating that cyclization does not significantly affect the protein structure. The proteins cz- 186 and were prepared, and used to isolate samples of the cz- 186/ complex, essentially as described previously (Hamdan et al., 2002a). NMR experiments made use of three different samples of complexes of unlabeled with isotope-labeled cz- 186: (i) a uniformly 13 C/ 15 N-labeled sample (0.5 mM), (ii) a biosynthetically directed fractional 13 C-labeled sample prepared from 20% 13 C-glucose (0.5 mM) (Neri et al., 1989, Senn et al., 1989), and (iii) a sample with 13 C/ 15 N-Leu (0.15 mM). Samples of 186/ were dialyzed against NMR buffer (20 mM Tris, pH 7.2, 100 mM NaCl, 0.1 mM dithiothreitol, and 0.08% (w/v) NaN3 in 90% H2O/10% D2O). Lanthanides (Ln 3+ = La 3+ or 1:1 mixtures of La 3+ /Dy 3+ or La 3+ /Yb 3+ ) were added from LnCl3 stock solutions in the same buffer containing total Ln 3+ concentrations of 30 mM. The 1:1 mixtures were added in slight molar excess to catalyze the metal ion exchange, resulting in exchange rates of a few s –1 (John et al., 2007a, John et al., 2007b).Restoration of the apo-complex was achieved by extensive dialysis against buffer containing 1 mM EDTA followed by dialysis against EDTA-free buffer. 2.3.2 NMR spectroscopy All NMR experiments were performed at 25 o C on a Bruker AV 800 MHz NMR spectrometer equipped with a cryogenic TCI probe. Sequence-specific resonance assignments of
2.3 Experimental section. 31 the methyl groups in the diamagnetic state were established by 3D HNCA and (H)CCH-TOCSY spectra of the uniformly 13 C/ 15 N labeled sample complexed with 1 equivalent of La 3+ (cz- 186/ /La 3+ ), and by reference to the assignments reported for the linear 186 protein with Mg 2+ (DeRose et al., 2003). Stereospecific assignments of Val and Leu methyl groups were obtained from a constant-time (28 ms) 13 C-HSQC spectrum recorded of the fractionally 13 C labeled sample. Where possible, the rotameric states of the side chains of Val and Leu residues in the crystal structure of 186 (Hamdan et al., 2002b) were confirmed in solution by a 3D NOESY- 15 N-HSQC spectrum (mixing time 60 ms) recorded of the uniformly 13 C/ 15 N labeled sample. Sequence-specific resonance assignments of the methyl groups in the paramagnetic state were established by 2D and 3D methyl Cz-EXSY spectra recorded with the pulse schemes of Figure 2.1 using a mixing period ( m) of 480 ms and spectral widths of 30 ppm ( 13 C) and 16 ppm ( 1 H). The 2D spectra were acquired with 160 × 1024 complex data points and 32 scans in 10 h, while the 3D spectra were acquired with 80 × 64 × 1024 complex points and 4 scans in 40 h. For all spectra, the initial t1 delay was set to half the increment so that folded paramagnetic peaks could be identified by their inverted sign (Bax et al., 1991). The methyl group assignments obtained with these experiments provided the controls for the assignment methods described below. Figure 2.1 Methyl CZ-EXSY experiments. (a, b) Pulse schemes of the 2D and 3D versions, respectively. Narrow and wide bars represent radiofrequency pulses with flip angles of 90º and 180º, respectively, applied with phase x unless indicated otherwise. Selective 13 C pulses were applied as a 1.5 ms Q5 pulse prior to the delay C and as a 1.5 ms time-reversed Q5
Page 1 and 2: Computational study of proteins wit
Page 3 and 4: iii Schmitz C, Stanton-Cook MJ, Su
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3.7 Study case. 87 The coordinates
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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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102 Chapter 4. Protein 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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