Introduction and overview


Representation of pseudocontact shift isosurfaces for a Tm3+-loaded lanthanide chelating tag conjugated to hCA (left). 1H-15N HSQC NMR spectra of 15N leucine labeled human Carbonic Anhydrase (hCA) tagged with different lanthanides: Dy: blue, Tm: red, Lu: black (right).

Nuclear magnetic resonance spectroscopy provides an ideal toolbox for elucidating structure, dynamics and interactions of biomacromolecules in solution, since the offered possibility to tune and adjust the experimental conditions, i.e. buffer components, salt concentration, additives, temperature and pH, yields realistic and atomic-resolution structures of biomacromolecules under physiological conditions. In order to study protein-protein or protein-ligand interactions as well as the positioning and conformation of flexible domains within proteins in solution, long-range structural restraints are urgently needed. Paramagnetic nuclear magnetic resonance spectroscopy, more specifically pseudocontact shifts and residual dipolar couplings generated by lanthanide chelating tags, can deliver such long-range restraints and thereby render amenable the structural analysis of large protein complexes as well as protein-ligand binding in solution.

"Design and application of lanthanide chelating tags for pseudocontact shift nuclear magnetic resonance spectroscopy on biomacromolecules."
D. Joss, D. Häussinger, Progress in Nuclear Magnetic Resonance Spectroscopy, 2019, 114-115, 284-312.


Design of next-generation lanthanide chelating tags

Tag Development

Overlay of DFT structures of Lu-P4T-DOTA and Lu-P4M4-DOTA and their structures (left, attachment point of the protein’s cysteine residue in magenta). Selection of induced shifts by the strongly paramagnetic Tm- and Dy-P4T-DOTA (right).

In order to generate strongly paramagnetic lanthanide chelating tags that are sufficiently immobilized on the surface of the protein and generate thereby large paramagnetic effects, i.e. large pseudocontact shifts and residual dipolar couplings, we synthesize sterically overcrowded DOTA-derived lanthanide chelating tags with different linker systems. More specifically, the introduction of sterically demanding substituents, e.g. isopropyl groups, as well as the introduction of novel, reduction-stable linker moieties, e.g. pyridinethiazole derivatives, proved to be highly beneficial in order to generate lanthanide chelating tags with optimal properties and general applicability.

"P4T-DOTA - A lanthanide chelating tag combining a highly sterically overcrowded backbone with a reductively stable linker."
D. Joss, D. Häussinger, Chemical Communications, 2019, 55, 10543-10546.

"A sterically overcrowded, isopropyl-substituted lanthanide chelating tag for protein PCS NMR spectroscopy: Synthesis of its macrocyclic scaffold and benchmarking on ubiquitin S57C and hCA II S166C."
D. Joss, M.-S. Bertrams, D. Häussinger, Chemistry - A European Journal, 2019, 25, 11910-11917.

"New Lanthanide chelating tags for PCS NMR spectroscopy with reduction stable, rigid linkers for fast and irreversible conjugation to proteins."
T. Müntener, J. Kottelat, A. Huber, D. Häussinger, Bioconjugate Chemistry, 2018, 29, 3344-3351.


Applications of lanthanide chelating tags

Isosurfaces hCA II

Tensors required for unambiguous localization of a given atom by using PCS as sole source of structural restraints. Top left: isosurface (red) and hCA II (grey). Top right: intersection of two isosurfaces. Bottom left: intersection of three isosurfaces resulting in two intersection points (one visible, one on the other side of the protein scaffold). Bottom right: intersection of four isosurfaces resulting in only one intersection point (visible in center).

The successful localization of ligands within biomacromolecules is crucial for drug development as well as the investigation of biological processes within cells. In order to unambiguously localize fluorinated ligands for the model protein human carbonic anhydrase II, we developed a pseudocontact shift based methodology that allows to obtain the position of the selected ligands over distances up to 38 Å with an accuracy of up to 0.8 Å within the protein scaffold.

"Localization of ligands within human carbonic anhydrase II using 19F pseudocontact shift analysis."
K. Zimmermann, D. Joss, T. Müntener, E. S. Nogueira, M. Schäfer, L. Knörr, F. W. Monnard, D. Häussinger, Chemical Science, 2019, 10, 5064-5072.

In Cell PCS and RDC

Introduction of tagged GB1 constructs into Xenopus laevis oocytes (left). Induced PCSs and RDCs in living cells at a field strength of 14.1 T (right).

Furthermore, it was shown for the first time that de novo protein structures can be determined within intact Xenopus laevis oocytes by analysis of pseudocontact shifts and residual dipolar couplings from a single set of 2D in-cell NMR experiments in combination with the Rosetta database. Thereby, structural ensembles of GB1 were conveniently derived from low concentration in-cell NMR samples (∼50 μM).  

"In-cell protein structures from 2D NMR experiments."
T. Müntener, D. Häussinger*, P. Selenko, F.-X. Theillet*, Journal of Physical Chemistry Letters, 2016, 7, 2821-2825.

SNARE complex PCS

Induced PCS visible in 1H-15N TROSY-HSQC spectra of a SNARE complex (left). Correlation plot of experimental and calculated PCS (middle). Isosurfaces induced by the lanthanide chelating tag incorporated into the complex of biomacromolecules (right). Blue: positive PCS, Red: negative PCS, Black: position of lanthanide ion. 

Besides the localization of ligands within biomacromolecules and the de novo structure determination of proteins in living cells, it was demonstrated by Brewer et al. that PCS contribute in valuable manner to the structure determination of large complexes of biomacromolecules. In the selected example, the researchers investigated the complex of biomacromolecules, i.e. Syt1 together with the SNARE complex (synaptobrevin, syntaxin-1 and SNAP-25), that is responsible for the rapid release of neurotransmitters.

"Dynamic Binding Mode of a Synaptotagmin-1-SNARE Complex in Solution."
K. Brewer, T. Bacaj, A. Cavalli, C. Camilloni, N. Barlow, A. Zhou, P. Cao, J. Xu, A. B. Seven, E. A. Prinslow, R. Voleti, D. Häussinger, A. Bonvin, J. Liu, D. R. Tomchick, M. Vendruscolo, B. Graham, T. C. Südhof, J. Rizo, Nature Structural & Molecular Biology, 2015, 22, 555-564.


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