Thesis Title: Subtitle - NMR Spectroscopy Research Group

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32 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. pulse prior to the delay H, generating an excitation bandwidth of 20 ppm. 1 H saturation is achieved with 120º pulses applied every 5 ms, and the 3-9-19 sequence is used for water suppression. During m, 180 o ( 1 H) pulses are applied every 6 ms with a MLEV-16 supercycle to suppress cross relaxation between 1 H and 13 C spins. The phase cycle was 1(x, –x), 2(2x, 2(–x)), 3(x), 4(4y, 4(–y)) and rec(x, 2(–x), x, –x, 2x, –x). States-TPPI was applied to 1 and 3 for quadrature detection. Delays: T = 28 ms, = 3 ms, C = 0.75 ms, H = 1.7 ms. Gradients (Gi) were applied along the z-axis with strengths of 23.2, 14.5, 20.3, 17.5 and 11.6 G/cm. (c-e) Simulated dipolar 13 C relaxation rates and NOE in isolated CH3 groups versus molecular rotational correlation time ( R) using eqs 1-4 in ref. 5. (c) Transverse relaxation rate R2, (d) longitudinal relaxation rate R1, and (e) steady-state 13 C{ 1 H} NOE. The dashed line reports the relaxation rates calculated for a static CH3 group, whereas the solid line takes into account a rapid rotation around the three-fold symmetry axis with a correlation time f = 25 ps and assuming tetrahedral geometry (Sf 2 = 0.111, rCH = 1.10 Å, rCC = 1.52 Å) (Wand et al., 1996). The dotted line represents the contribution from a neighboring 13 C spin to R2, R1, and cross-relaxation ( ), respectively. The vertical axis of the 13 C- 13 C cross- relaxation rate in (e) is in s –1 . Due to the small contribution of PRE to R1 (John et al., 2007a), the 13 C{ 1 H} NOE is similar for paramagnetic and diamagnetic proteins. 2.3.3 Manual resonance assignments from PCS The PCS measured from EXSY spectra were used to evaluate the possibility of assigning the methyl peaks by comparison with back-calculated PCS. PCS were back-calculated using a Mathematica (Wolfram <strong>Research</strong>) script and the crystal structure of 186 (PDB entry 1J53, ref. 40). The -tensor parameters of Dy 3+ in complex with 186/ have been reported previously (Schmitz et al., 2006). The tensor parameters for Yb 3+ were determined from 15 N-HSQC spectra using the program Echidna (Schmitz et al., 2006) as: ax = –6.52 × 10 –32 m 3 , rh = 1.12 × 10 –32 m 3 , = 24.4º, = 84.5º, and = –299.5º (using the zxz convention of Euler angle rotations). 1 H PCS of methyl groups were calculated for each of the three methyl protons individually and averaged. This average is largely insensitive to the rotational position of the methyl group. Residual CSA effects due to paramagnetic alignment (John et al., 2005) were disregarded since CSA tensors of methyl groups are small (Liu et al., 2003). 2.3.4 The program Possum
2.3 Experimental section. 33 The program Possum (paramagnetically orchestrated spectral solver of unassigned methyls) was developed to assign the cross-peaks of methyl groups in correlation spectra recorded with diamagnetic and paramagnetic metal ions by reference to the 3D structure of the protein and independently determined tensors. The program requires that the amino-acid type is known (e.g. by residue-type selective 13 C-labeling). Furthermore, it can accept information about methyl cross- peaks belonging to the same residue (―methyl connectivity‖ data for Ile, Leu, and Val, as provided by HCCH-TOCSY experiments) and stereospecific information (―methyl specificity‖ data distinguishing between 2 and 1 cross-peaks of Ile, 1 and 2 cross-peaks of Leu, and 1 and 2 cross-peaks of Val, as provided by samples produced with biosynthetically directed fractional 13 C- labeling (Neri et al., 1989, Senn et al., 1989, Tugarinov et al., 2004) or stereoselective isotope labeling (Ostler et al., 1993, Kainosho et al., 2006)). In the present version of the program, the methyl connectivity information is always assumed to be available for the diamagnetic state. The program takes as input the 1 H and 13 C chemical shifts of methyl groups measured in 13 C-HSQC spectra and the 13 C chemical shifts of methyl groups that are too close to the paramagnetic center to be directly observable in 1 H-detected <strong>NMR</strong> spectra. By comparing the chemical shifts in the diamagnetic and paramagnetic states, Possum attempts to find the resonance assignment with the lowest residual cost C(l) defined by: with: subject to: (2.1) (2.2)
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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3.6 Program Features. 83 A new PCS
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3.6 Program Features. 85 Carlo prot
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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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