P4T-DOTA – a lanthanide chelating tag combining a sterically highly overcrowded backbone with a reductively stable linker

Abstract

A highly rigidified lanthanide complex induces strong pseudocontact shifts and residual dipolar couplings for structural analysis of proteins in solution.

NMR pseudocontact shifts in a symmetric protein homotrimer

“…LCTs based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) framework provide large PCSs and are usually attached to the protein surface via one or two cysteine residues (Joss and Häussinger 2019a ; Nitsche and Otting 2017 ; Keizers et al 2007 ; Prudencio et al 2004 ). Recent developments of LCTs with reduction-stable linkages have enabled studies under physiologically relevant conditions in living cells (Müntener et al 2016 , 2018 ; Pan et al 2016 ; Joss and Häussinger 2019b ; Liu et al 2014 ; Hikone et al 2016 ). Nevertheless, many soluble proteins, as well as membrane proteins, form homomultimeric complexes (Hashimoto et al 2011 ; Ali and Imperiali 2005 ; Goodsell and Olson 2000 ), which are not readily accessible by PCS NMR spectroscopy due to formation of multi-tagged protein complexes.…”

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

“…When compared to other LCTs, the anisotropy parameters induced by Ln-M7-Nitro are exceedingly large and the value for the Dy-loaded chelator attached to ubiquitin S57C constitutes an unprecedented value of Dw ax for an LCT-protein construct. 17,19,20,22,29 The obtained Q-factors and the distance of the fitted lanthanoid metal centre to the C beta -carbon of the cysteine residue (6.9 Å) indicate excellent fits. Notably, RDCs up to 41.3 Hz were obtained for the Dy-loaded chelator, while RDCs of up to 35.9 Hz were obtained using the Tb complex (Tables S11 and S12, ESI †).…”

“…5,6 Conformational and positional invariant LCTs are ideally suited for monitoring protein interactions, as they can be placed far away from the interface and nevertheless provide accurate spatial information up to 200 Å. Earlier approaches to suppress the translational and rotational freedom of the chelator on the surface of the protein, and hence to induce long-range restraints in solution, include: (1) two linker moieties attached to a double cysteine mutant, [17][18][19] (2) restriction of the translational and rotational mobility, [20][21][22] (3) placement of bulky groups on the macrocyclic ring scaffold. 22,23 In order to restrict the remaining rotational averaging by para-substituted linker moieties, 20,21 we rationally designed a novel, ortho-substituted pyridine activator that leads to a hampered rotation around the S cysteine -C pyridine bond and to a short, reduction-stable linkage between the LCT and the biomacromolecule ( Fig.…”

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

“…In order to provide more rigid chelators and attach an LCT at any possible position within the scaffold of the biomacromolecule, a large variety of double-and single-armed lanthanoid chelating tags (LCTs) has been developed. [28][29][30][31][32][33][34][35][36][37] LCTs have since then found widespread applications in structural biology and biomolecular NMR, e.g. structural characterization of antibiotic drug targets (Fig.…”