Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules

Abstract

In this review, lanthanide chelating tags and their application in pseudocontact shift NMR spectroscopy as well as analysis of residual dipolar couplings are covered. Including a complete overview of non-DOTA-derived and DOTAderived lanthanide chelating tags, critical points in the design of lanthanide chelating tags as appropriate linker moieties, stability under reductive conditions e.g. for in-cell applications, magnitude of the transferred anisotropy from the lanthanide chelating tag to the biomacromolecule under investigation and structural properties as well as conformational bias of the lanthanide chelating tags are discussed. Furthermore, all currently published DOTAderived lanthanide chelating tags used for PCS NMR spectroscopy are displayed in tabular form including their anisotropy parameters with all employed lanthanide ions, CB-Ln distances and tagging reaction conditions, i.e. stoichiometry of lanthanide chelating tag, pH, buffer composition, temperature and reaction time. Additionally, reported applications of lanthanide chelating tags for pseudocontact shifts and residual dipolar couplings on proteins, protein-protein and protein-ligand complexes, carbohydrates, carbohydrate-protein complexes, nucleic acids and nucleic acid-protein complexes are presented and critically reviewed. The vast and impressing field of applications of lanthanide chelating tags in structural investigations of biomacromolecules in solution clearly illustrates the significance of this particular field of research and the extension of the repertoire of lanthanide chelating tags from proteins to nucleic acids holds great promise for the determination of valuable structural parameters and further developments in characterising intermolecular interactions.

NMR pseudocontact shifts in a symmetric protein homotrimer

“…While residual dipolar couplings (RDCs) determine relative orientations of bond vectors (Chen and Tjandra 2012 ), nuclear Overhauser effects (NOEs) provide short-range distance information (Wagner and Wüthrich 1982 ; Williamson 2009 ). On the other hand, the two paramagnetic effects paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS), which can be introduced into proteins by various lanthanide-chelating tags (LCTs), have proven to provide valuable distance- and orientation-dependent information over extremely long distances exceeding 100 Å (Joss and Häussinger 2019a ; Nitsche and Otting 2017 ; Wang et al 2007 ; Bertini et al 2002a , b ; Gochin and Roder 1995 ; Pearce et al 2017 ). 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 ).…”

“…On the other hand, the two paramagnetic effects paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS), which can be introduced into proteins by various lanthanide-chelating tags (LCTs), have proven to provide valuable distance- and orientation-dependent information over extremely long distances exceeding 100 Å (Joss and Häussinger 2019a ; Nitsche and Otting 2017 ; Wang et al 2007 ; Bertini et al 2002a , b ; Gochin and Roder 1995 ; Pearce et al 2017 ). 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 ).…”

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

“…lanthanoid ions complexed with suitable chelators for attachment, provide a unique and precise tool for the study of the properties of biomacromolecules by solution 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. 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.…”

“…The precursor of the LCT with all four side arms installed (3) was then deprotected by heating of the compound with hydrochloric acid in acetonitrile to give the free ligand 4. Subsequently, the chelator was loaded with lanthanoid ions suitable for the investigation of paramagnetic effects on biomacromolecules (5) and the leaving group was activated by oxidation of the thioether to the methylsulphone (6).…”