New Lanthanide Chelating Tags for PCS NMR Spectroscopy with Reduction Stable, Rigid Linkers for Fast and Irreversible Conjugation to Proteins

Abstract

Lanthanide chelating tags (LCTs) have been used with great success for determining structures and interactions of proteins and other biological macromolecules. Recently LCTs have also been used for in-cell NMR spectroscopy, but the bottleneck especially for demanding applications like pseudocontact shift (PCS) NMR is the sparse availability of suitable tags that allow for site-selective, rigid, irreversible, fast, and quantitative conjugation of chelated paramagnetic lanthanide ions to proteins via reduction stable bonds. We report here several such tags and focus on a new pyridine thiazole derivate of DOTA, that combines high affinity, rigidity, and selectivity with unprecedented tagging properties. The conjugation to the cysteine thiol of the protein results in a reductively stable thioether bond and proceeds virtually quantitatively in less than 30 min at 100 μM protein concentration, ambient temperature, and neutral pH. Upon conjugation of the new tag to two single cysteine mutants of ubiquitin and a single cysteine mutant of human carbonic anhydrase type II (30 kDa) only one stereoisomer is formed (square antiprismatic coordination, Λ(δδδδ)) and large to very large pseudocontact shifts as well as large residual dipolar couplings (RDCs) are observed by NMR spectroscopy. The PCS and RDC show excellent agreement with the solid state structure of the proteins. We believe that the pyridine thiazole moiety reported here has the potential to serve as a thiole reactive group in various conjugation applications; furthermore, its terbium complex shows strong photoluminescence upon irradiation and may thus serve as a donor group for Förster resonance energy transfer spectroscopy.

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 tag showed PCS of up to 6.5 ppm when attached to ubiquitin S57 C, and the PCS and tensor anisotropies observed for Ln‐P4M4‐DOTA upon attachment to the protein were significantly increased compared to those of its methylated predecessor (Figures and and ; Table ). The magnitude of the obtained PCS and anisotropy parameters as well as the straightforward application places Ln‐P4M4‐DOTA among the best performing LCTs currently available …”

“…The magnitude of the obtained PCS and anisotropy parameters as well as the straightforwardapplication places Ln-P4M4-DOTAamong the best performing LCTscurrently available. [3,9,11,17,19,30,31] The enhanced PCS could be attributedt oo ne or more of the following factors:( i) decreased small-amplitude vibrations of the scaffold,( ii)different positioning of the oxygen donor atomso ft he side arms relative to the nitrogen donors of the basal cyclen,w hich was shown by Mironov et al [32] to significantly affect the observed anisotropies, (iii)alteredm obility or rotationoft he tag on the protein surface. As can be seen from the calculated structure of the Lu-P4M4-DOTAc omplex, the isopropyl groupso nt he backbone lead to extreme overcrowding of the chelating cage ( Figure 10).…”

“…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.…”

The Photocatalyzed Thiol‐ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins

“…The standard strategy is the conjugation by formation of a disulfide bond: this technique has been widely used but it is hampered by the stability of the bond formed . Thus, in recent years, much effort has been concentrated on the design of paramagnetic tags that are stable under reducing conditions once attached to the protein, and which could also be used in cells . For this purpose, copper‐catalyzed azide−alkyne “click” cycloadditions, (which, however, require the introduction of non‐natural amino acids i. e. p‐azido‐phenylalanine in the protein sequence), and thiol‐ene coupling (TEC) (which relies on existing or mutated cysteine residues) have emerged as useful alternative tools in protein chemical conjugation.…”