Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags

A Sterically Overcrowded, Isopropyl‐Substituted, Lanthanide‐Chelating Tag for Protein Pseudocontact Shift NMR Spectroscopy: Synthesis of its Macrocyclic Scaffold and Benchmarking on Ubiquitin S57 C and hCA II S166 C

“…The tensor properties were then obtained by fitting to the residues in secondary structure elements of ubiquitin (PDB 1UBI 37 ) or the leucine residues of hCA II (PDB 3KS3) using Numbat . Q‐factors were calculated according to the standard equation described by Nitsche et al …”

“…[33,39,40] The tensor properties were then obtained by fitting to the residues in secondary structure elements of ubiquitin (PDB 1UBI 37 )o rt he leucine residues of hCA II (PDB 3KS3 [41] )u sing Numbat. [42] Q-factors were calculated according to the standard equation described by Nitsche et al [1] DFT calculations DFT calculations were performed with the ORCA program package [43] at the sciCORE facility of the University of Basel. For these calculations, BP86 was used as the functional, [44,45] SARC-TZVP as the basis set for the ligands, and SARC2-QZVP as the basis set for the lanthanide metal.…”

“…Pseudocontact shifts (PCS) and residual dipolar couplings (RDC) generated by lanthanide‐chelating tags (LCT) yield valuable structural restraints for the analysis of structure, dynamics, and ligand‐binding of proteins in solution . In order to access valuable structural restraints by PCS measurements, stereospecifically methyl‐substituted 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA)‐based chelators present a suitable scaffold .…”

“…Pseudocontact shifts (PCS) and residual dipolar couplings (RDC) generated by lanthanide-chelatingt ags (LCT) yield valuable structural restraints for the analysis of structure, dynamics, and ligand-binding of proteins in solution. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] In order to access valuable structuralr estraintsb yP CS measurements, stereospecifically methyl-substituted1 ,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-based chelators present a suitable scaffold. [9,[19][20][21][22] As investigated by Ranganathane tal., the methyl substituents on the nitrogen-containingm acrocycle in al anthanide complex adopt an equatorial-upperp osition, [23,24] that is, the substituents pointa way from the metal centre in order to accommodate the metal ion in the ligand cavity.I norder to obtain only one signal set in 1 H- 15 NHSQC experiments and to induces trong paramagnetic effects fort he investigation of biomacromolecules in solution, it is mandatory to lock not only the 12-membered ring, [9] but also the pendant arms of the chelator,i no ne conformation.A ss hown by Joss et al,T m-and Dy-DOTA-M8-(4R,4S)-SSPy adopt a L(dddd)c onformation,r esulting in as ingle set of peaks in 1 H- 15 NH SQC spectra.…”

Protein Fold Determination by Assembling Extended Super-Secondary Structure Motifs Using Limited NMR Data

“…The DINGO-PCS utilizes PCSs of backbone amide protons as the only experimental data to identify, orient, and build the protein structure from its constituent Smotifs. The DINGO-PCS algorithm requires PCS datasets to be available from at least three different metal centers that are site-specifically incorporated in the target protein using artificial metal carrying chemical tags (Nitsche and Otting, 2017). The locations of these metal centers are ideally well dispersed in space to reduce correlation between PCS data sets, which can be difficult to achieve without prior knowledge of the target structure.…”