Thesis Title: Subtitle - NMR Spectroscopy Research Group

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76 Chapter 3. Numbat: new user-friendly method built for automatic Δχ-tensor determination. 3.1 Abstract Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in <strong>NMR</strong> spectroscopy as they yield a complementary set of orientational and long-range structural restraints. PCS are a manifestation of the χ-tensor anisotropy, the Δχ-tensor, which in turn can be determined from the PCS. Once the Δχ-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein-ligand complexes. Here we present the newly developed package Numbat (New User- friendly Method Built for Automatic Δχ-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Δχ-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits ε186 and θ using PCS. 3.2 Keywords paramagnetic <strong>NMR</strong> · pseudocontact shift · magnetic susceptibility tensor · software · program · unique tensor representation 3.3 Abbreviations α Subunit α of the E. coli polymerase III ε186 N-terminal 185 residues of the E. coli polymerase III subunit ε θ Subunit θ of the E. coli polymerase III CSA Chemical shielding anisotropy GUI Graphical user interface HOT The bacteriophage P1-encoded homolog of θ PCS Pseudocontact shift RACS Residual anisotropic chemical shift RDC Residual dipolar coupling UTR Unique Δχ-tensor representation
3.4 Introduction 3.4 Introduction. 77 Paramagnetic lanthanide ions bound to the natural metal-binding site of a metalloprotein or introduced via a lanthanide tag provide a number of paramagnetic effects that can be distance dependent (i.e. paramagnetic relaxation enhancement), orientation dependent (i.e. residual dipolar couplings, RDC), or a combination of both, like cross-correlated relaxation effects and pseudocontact shifts (PCS;(Bertini et al., 2002, Pintacuda et al., 2004)). PCS present particularly valuable structural restraints, as they are easy to measure and provide long-range information that would be difficult to obtain by other techniques. PCS originate from unpaired electron spins which lead to an anisotropic magnetic susceptibility tensor (χ-tensor). PCS restraints induced by lanthanide ions have been used to investigate structural and dynamical properties of proteins (Allegrozzi et al., 2000, Bertini et al., 2001, Bertini et al., 2004, Gaponenko et al., 2004, Jensen et al., 2006, Eichmüller et al., 2007, Wang et al., 2007) and protein-ligand complexes (John et al., 2006, Pintacuda et al., 2007). In order to apply PCS restraints, eight variables have to be determined. These comprise the lanthanide position (three Cartesian coordinates), three angles (e.g. Euler angles) that relate the molecular frame to the χ-tensor frame, and the axial and rhombic anisotropy parameters of the χ- tensor. (Since PCS depend only on the χ-tensor anisotropy Δχ rather than the absolute magnitude of the χ-tensor, it is sufficient to determine the anisotropy parameters represented by the Δχ-tensor.) Several integrated software tools are available for the determination and study of the alignment tensor using RDCs (Dosset et al., 2000, Zweckstetter et al., 2000, Valafar et al., 2004, Wei et al., 2006). For the situation where the 3D structure of the protein is known a priori, corresponding tools for the determination of the Δχ-tensor from PCS have been developed but are more limited in scope. The program Fantasia (Banci et al., 1996) and its extension Fantasian (Banci et al., 1997) can fit the magnitude and Euler angles of the Δχ-tensor using a set of experimental PCS but requires prior knowledge of the metal coordinates. The program Platypus (Pintacuda et al., 2004) can simultaneously fit the Δχ-tensor and assign the signals of 15 N-HSQC spectra of samples containing diamagnetic and paramagnetic lanthanides, but assumes that the 15 N-HSQC peaks are sufficiently well resolved such that the paramagnetic peaks can be unambiguously associated with their diamagnetic partners. The program Echidna (Schmitz et al., 2006) uses assigned diamagnetic 15 N-HSQC cross-peaks of a uniformly 15 N-labelled protein to determine the magnitude and Euler angles of the Δχ- tensor and, simultaneously, the assignment of the paramagnetic 15 N-HSQC cross- peaks. It also requires prior knowledge of the approximate metal ion position. In principle, the structure refinement packages Xplor-NIH (Schwieters et al., 2003, Schwieters et al., 2006) with the
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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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vii Keywords paramagnetic nmr, pseu
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ix 2.3.3 Manual resonance assignmen
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xi List of Figures Figure 1.1 NMR e
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xv List of Abbreviations α Subunit
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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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