Thesis Title: Subtitle - NMR Spectroscopy Research Group

More documents

Recommendations

Info

44 Chapter 2. Possum: paramagnetically orchestrated spectral solver of unassigned methyls. (Figure 2.5d). A rare exception is Val50, where the predicted 13 C and 1 H PCS of the 1 and 2 methyl groups are indistinguishable in both the Dy 3+ and Yb 3+ complex. The methyl groups of Ile residues are particularly easy to assign by PCS, since the spectral ranges of the 13 C-<strong>NMR</strong> signals of 2 and 1 methyl groups are clearly separated, while intra- residual methyl-methyl connectivity can still be obtained from TOCSY spectra. 2.4.5 Automatic assignments without EXSY data Cz-EXSY spectra provide an exceptionally simple way of measuring PCS. For situations where the metal exchange is too slow for exchange spectra and spectral crowding prevents the straightforward pairing between diamagnetic and paramagnetic 13 C-HSQC peaks (Figure 2.3), we have devised the program Possum which determines the correct peak pairings, their resonance assignments, and their PCS, using the 3D structure of the protein and the tensor (that can readily be obtained from, e.g. , 15 N-HSQC spectra (Pintacuda et al., 2004, Schmitz et al., 2006)). The performance of the program was initially tested with simulated data, replacing the experimental paramagnetic shifts of Table S2.1 by shifts back-calculated from the crystal structure of 186 (Hamdan et al., 2002b) and using the crystal structure, the experimental diamagnetic chemical shifts, and the tensors of Dy 3+ and Yb 3+ as input. In all calculations, it was assumed that the residue types of all methyl resonances were known and the methyl connectivity information of Val, Leu, and Ile residues was available for the diamagnetic state. Except for extreme cases of spectral overlap, the program yielded 100% correct assignments. In a second step, structural uncertainties were simulated by randomly displacing the methyl groups, following a Maxwell- Boltzmann distribution with maxima at 0.35 and 0.7 Å (resulting in maximal atom displacements of 0.75 and 1.5 Å, respectively, always using the same direction of displacement). Even in the case with the maximum structural noise, using only paramagnetic data from the Yb 3+ complex and neither methyl specificity nor methyl connectivity information in the paramagnetic state, Possum yielded >75% correct assignments of the diamagnetic methyl resonances (Table S2.2 and Table S2.4). The score increased to >90% when paramagnetic data from the Dy 3+ complex, methyl specificity information in all complexes, and methyl connectivity information in the Yb 3+ complex (but not the Dy 3+ complex) were included (Table S2.2 and Table S2.3). The program was subsequently applied to the experimental data of the methyl groups of cz- 186/ loaded with La 3+ , Yb 3+ and Dy 3+ . Table 2.1 summarizes the results. Using both paramagnetic lanthanides, the assignment is complete and correct for all diamagnetic 13 C-HSQC
2.4 Results. 45 cross-peaks that have observable paramagnetic partners. The only exceptions are swapped assignments for Met18 and Met107 and Val65 and Val82. The first arises from a side-chain conformation that is different in solution than in the single crystal and the second from differences in the predicted and experimental PCS observed for the peptide segment near Val65 (John et al., 2007b).The assignments of the methyl groups of the Yb 3+ complex are similarly reliable, whereas the methyl signals of the Dy 3+ complex are harder to assign (in the absence of methyl connectivity information). Using only data from the Yb 3+ complex and omitting any methyl specificity information or connectivity information in the paramagnetic state still results in >70% correct assignments of the diamagnetic methyl resonances (Table S2.2 and Table S2.4) 4 . Table 2.1 Automatic assignment of methyl groups by the program Possum a Residue type Occurrence b La c observable Yb d assigned Dy d assigned La e assigned Met 6 (1) 5 3/5 4/4 3/5 Thr 14 (4) 8 7/7 7/7 7/7 Ala 19 (2) 17 13/13 11/13 14/14 Ile 14 (2) 24 24/24 21/23 24/24 Val 12 (0) 24 20/20 17/20 18/22 Leu 17 (0) 34 34/34 19/25 34/34 a Obtained using the data reported in Table S2.1, the crystal structure of 186 (Hamdan et al., 2002b) and tensors determined from 15 N-HSQC spectra as described in the experimental section. The paramagnetic data measured with Yb 3+ and Dy 3+ were combined to derive the assignments. b Total number of residues in cz- 186. The number in brackets refers to residues not observed in the crystal structure; these were excluded from the calculation. c Number of methyl groups with coordinates reported in the crystal structure for which cross-peaks were observed in the cz- 186/ /Ln 3+ sample. Their unassigned chemical shifts were available for the program. 4 It is the responsibility of the user to inspect and optimize the assignment provided by Possum; in particular to untangle areas of the spectrum with overlapping peaks.
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
Page 13 and 14: xiii
Page 15: xv List of Abbreviations α Subunit
Page 18 and 19: 2 Chapter 1. Introduction. The scal
Page 20 and 21: 4 Chapter 1. Introduction. In contr
Page 22 and 23: 6 Chapter 1. Introduction. as the c
Page 24 and 25: 8 Chapter 1. Introduction. Figure 1
Page 26 and 27: 10 Chapter 1. Introduction. Protein
Page 28 and 29: 12 Chapter 1. Introduction. 1.3 Com
Page 30 and 31: 14 Chapter 1. Introduction. Figure
Page 32 and 33: 16 Chapter 1. Introduction. Figure
Page 34 and 35: 18 Chapter 1. Introduction. (Bugaye
Page 36 and 37: 20 Chapter 1. Introduction. The det
Page 38 and 39: 22 Chapter 1. Introduction. (ii) It
Page 40 and 41: 24 Chapter 1. Introduction. Chapter
Page 42 and 43: 26 Chapter 1. Introduction. Su XC,
Page 44 and 45: 28 Chapter 2. Possum: paramagnetica
Page 91 and 92: Chapter 3 Numbat: new user-friendly
Page 93 and 94: 3.4 Introduction 3.4 Introduction.
Page 95 and 96: 3.5 Algorithm. 79 rhombic component
Page 97 and 98: 3.6 Program Features. 81 each fitti
Page 99 and 100: 3.6 Program Features. 83 A new PCS
Page 101 and 102: 3.6 Program Features. 85 Carlo prot
Page 103 and 104: 3.7 Study case. 87 The coordinates
Page 105 and 106: 3.7 Study case. 89 illustrates how
Page 107 and 108: 3.7 Study case. 91 can be used to a
Page 109 and 110: 3.9 Acknowledgment. 93 variables, t
Page 111 and 112:
3.10 References. 95 Galassi M, Davi
Page 113 and 114:
3.11 Supporting information. 97 Zwe
Page 115:
3.11 Supporting information. 99 Tab
Page 118 and 119:
102 Chapter 4. Protein Structure De
Page 120 and 121:
Page 122 and 123:
Page 124 and 125:
Page 126 and 127:
Page 128 and 129:
Page 130 and 131:
Page 132 and 133:
Page 134 and 135:
Page 136 and 137:
Page 138 and 139:
Page 140 and 141:
Page 142 and 143:
Page 145 and 146:
Chapter 5 Conclusion and perspectiv
Page 147 and 148:
5.1 The use of PCS for structure de
Page 149 and 150:
5.1 The use of PCS for structure de
Page 151 and 152:
5.3 The use of PCS for protein dock
show all

Thesis Title: Subtitle - NMR Spectroscopy Research Group

Create successful ePaper yourself

Delete template?

Save as template?