Application of Paramagnetic Lanthanoid Chelating Tags in NMR Spectroscopy and Their Use for the Localization of Ligands Within Biomacromolecules

Abstract

DOTA-Based Lanthanoid Chelating Tags 8 3 Applications of Lanthanoid Chelating Tags in the Localization of Ligands Within Biomacromolecules 12 3.1 Introduction 12 3.2 Weakly Binding Ligands 12 3.3 Ligands in the Intermediate Exchange Regime 13 3.4 Strongly Binding Ligands 14 4 Localization of Ligands Within Human Carbonic Anhydrase II Using 19 F Pseudocontact Shift Analysis 15 4.1 Human Carbonic Anhydrase and Its Use in the Development of Artificial Metalloenzymes 15 4.2 Localization of Ligands within Human Carbonic Anhydrase II-Introduction and Concept 16 4.3 PCS Analysis and Determination of the Anisotropy Parameters from Multiple Positions Within the Protein 18 4.4 Measurement of 19 F PCS and Localization of the Ligand Using a Monte-Carlo Approach 19 4.5 Determination of the Ligand Position by Analysis of the Isosurfaces of the Anisotropy Parameters 24 4.6 1 H-19 F-HOESY Experiments for Determination of 1 H PCS of the Ligand and Protein-Ligand Interactions 24 5 Conclusion and Outlook 26 Acknowledgment 26 References 26

A novel, rationally designed lanthanoid chelating tag delivers large paramagnetic structural restraints for biomolecular NMR

“…[3][4][5][6] More specifically, lanthanoid chelating tags (LCT) induce longrange restraints that provide the unique opportunity to elucidate the structure of complexes, dynamics and interactions of biomacromolecules and ligands. [7][8][9][10][11][12][13][14][15][16] In order to induce large paramagnetic effects, i.e. large pseudocontact shifts (PCS), residual dipolar couplings (RDC) and paramagnetic relaxation enhancement (PRE), rigid immobilization of the lanthanoid chelator on the surface of the biomacromolecule of interest is crucial.…”

“…In order to test the magnitude of the induced PCS and RDC effects of Ln-M7-Nitro on ubiquitin S57C and selectively 15 N leucine labelled hCA II S50C protein constructs, paramagnetic Tm-, Dy-, Tb-and Yb-loaded complexes were conjugated to the Table 1 Induced axial and rhombic components of the paramagnetic susceptibility tensors (Dw ax and Dw rh , in 10 À32 m 3 ), metal position in PDB coordinate frame (X metal , Y metal , Z metal , in Å), Euler angles (a, b, g, in 1) and quality factor (Q, mathematical definition in ESI, p. 1) on ubiquitin S57C (pH 6.0) and selectively 15 N leucine labelled hCA II S50C (pH 6.8) at 298 K. PCS data was fitted using Numbat, 32 while RDC tensors were obtained with Fanten. 33 A detailed table with single and joint fits of PCS/RDC is available in the ESI (Tables S17 and S18)…”

“…PRE is an isotropic effect that enhances the relaxation of spins in vicinity to the paramagnetic metal centre and causes thereby line broadening, which can be quantified by plotting the intensity ratio of paramagnetic and diamagnetic peaks vs. the distance of the amide proton to the metal centre. Upon attachment of the Gd complex of the newly developed LCT to both ubiquitin S57C and hCA II S50C, strong PREs were detected in 1 H- 15 N HSQC experiments and evaluated with respect to their distance dependence, the amino acid sequence of the protein and the theoretically predicted values for an LCT-protein construct with the same molecular weight (Fig. S9-S12, ESI †).…”

“…Anisotropy parameters induced by Ln-M7-Nitro on ubiquitin S57C (top) and selectively15 N leucine labelled hCA II S50C (bottom) depicted as isosurfaces with a given shift in ppm (generated using Numbat32 ). Top (from left to right): Tm-, Dy-and Tb-M7-Nitro-Ub S57C (inner layer: 4.0 ppm, outer layer: 1.5 ppm).…”