Research
Isotope Labeling for Biomolecular NMR
The molecular structure of proteins dictates the spatial distribution of steric and electronic interaction sites, which are critical for the function and regulation of essential cellular processes. Alterations in conformational properties associated with pathogenic cell states contribute to the onset and progression of diseases such as cancer, autoimmune disorders, and neuropathological conditions. Effective optimization of existing therapeutics and the design of novel drug scaffolds require precise knowledge of the structural parameters of the corresponding targets. The rapid development of biomolecular NMR spectroscopy in the last decade(s) has paved the way for novel insights into the structure, dynamic properties, as well as the interaction of proteins. However, NMR data interpretation of large protein complexes is only feasible if the corresponding sample is selectively enriched in NMR active nuclei (13C and 15N) and/or 1H-depleted by 2H incorporation. One important strategy for selective protein isotope labeling is given by the addition of metabolic amino acid precursor compounds to the growth medium of a protein-overexpressing microorganism.
Synthesis of Selectively 13C/2H/15N-Labeled Arginine to Probe Protein Conformation and Interaction by NMR Spectroscopy
Chem. Eur. J. 2025, 31, 24, e202500408
Darja I. Rohden, Giorgia Toscano, Paul Schanda, Roman J. Lichtenecker
Together with the Schanda Group (IST Austria), we developed a new labeling approach to introduce isolated 15N−13C−1H spin systems to the side chains of arginine residues. The labeling scheme delivers high-resolution NMR spectra to study large protein complexes. The interaction between ubiquitin and the third Src homology 3 domain of yeast protein Sla1 (Sla1 SH3-3) has been analyzed, highlighting the probes’ competence to sample rapid dynamics. Unlike the case of methyl-labeling strategies, the method involves the synthesis of the amino acid instead of a metabolic precursor, which expands its application beyond bacterial overexpression to the use in cell-free protein synthesis or eukaryotic protein production.
The synthesis of specifically isotope labelled Fluorotryptophan and its use in mammalian cell-based protein expression for 19F-NMR applications.
Chem. Comm. 2024, 60, 14188.
Giorgia Toscano, Martina Rosati, Letizia Barbieri, Katharina Maier, Lucia Banci, Enrico Luchinat, Robert Konrat, Roman J. Lichtenecker
19F nuclei serve as versatile sensors for detecting protein interactions and dynamics in biomolecular NMR spectroscopy. Although various methods have been developed to incorporate fluorine-containing aromatic residues into proteins using E. coli or cell-free expression techniques, similar approaches for protein production in mammalian cell lines remain limited. Together with our collaborators from the University of Florence we present a cost-effective synthetic route to obtain selectively deuterated, carbon-13 labeled fluorotryptophan and demonstrate its use in introducing 19F–13C spin pairs into carbonic anhydrase 2 and superoxide dismutase, following an expression protocol utilizing HEK cells.
Synthesis of a 13C/2H Labeled Building Block to Probe the Phosphotyrosine Interactome Using Biomolecular NMR Spectroscopy
ChemBioChem 2025, 26, 1, e202400663
Sarah Kratzwald, Thomas C. Schwarz, Karin Ledolter, Matus Hlavac, Manuel Felkl, Christian F. W. Becker, Robert Konrat, Dr. Roman J. Lichtenecker
A protected 13C/2H-labeled phosphotyrosine (pTyr) building block was synthesized from commercially available isotope sources for use in solid-phase peptide synthesis (SPPS). The resulting isolated ϵ-aromatic 13C-1H correlations provide valuable data for studying interactions with pTyr-binding proteins at atomic resolution using NMR spectroscopy techniques. As an initial demonstration, intermolecular NOE and chemical shift perturbation data were recorded to visualize the binding of a pTyr-containing peptide, derived from the platelet-derived growth factor to the PLCγ-1 SH2 domain.
A Compound Toolbox for Tailor-Made Isotope Labelling
Throughout the last years, the group developed several new compounds for selectively introducing C-13, N-15, F-19 and deuterium into target proteins. Highlights include the separation of Valine and Leucine labelling in E. coli based overexpression, the introduction of alpha ketoacid precursors for Phenylalanine and Tyrosine Labelling, novel metabolic intermediates for Tryptophan and Histidine labelling, as well as economic synthetic approaches to access isotopologues of Arginine and Proline. Additionally, we introduced methods to create 13C-F spin systems in various aromatic side-chain positions.
