Biomolecular NMR Spectroscopy with Paramagnetic Labels

Paramagnetic metal ions (e.g. Mn2+, Cu2+, Co2+, lanthanides) offer outstanding opportunities for the study of biomacromolecules by NMR spectroscopy. The paramagnetic NMR effects can be observed at large distances (up to 40 Å) from the paramagnetic center and provide valuable restraints for the determination of the three-dimensional structure of proteins and their interactions with other biomolecules or small molecule ligands. The development of novel metal ion-binding tags and strategies for their covalent attachment to proteins have broadened the applicability of paramagnetic NMR, so that proteins without a natural metal binding site can also be studied.

Paramagnetic NMR effects manifested in the NMR spectrum provide highly valuable restraints for structural modeling. (A) NMR spectrum of IL-10 in the presence of dia- or paramagnetic lanthanides. PCS vectors of selected residues are indicated as black lines. (B) Left: Cartoon representation of IL-10. The PCSs induced by Tb3+ are depicted as isorsurfaces. Right: Model for the interaction of IL-10 with heparin obtained by PCS-guided docking. (C) Schema depicting the NMR data that are accessible through ROSETTA.

Our research group develops and applies paramagnetic NMR methods for the structure elucidation of proteins and the study of protein-ligand interactions. In the past, we could determine structural models of the membrane protein DsbB and of the soluble protein IL-10 bound to glycosaminoglycans using paramagnetic NMR experiments. We have also developed a computational framework for the Rosetta software which integrates different paramagnetic NMR restraints (PCSs, RDCs, PREs) and combines them with other local NMR data (chemical shifts, NOEs). The RosettaNMR frameworks supports several modeling scenarios, including de novo protein folding, protein docking, ligand docking, and modeling of symmetric complexes. Recently, we have demonstrated an NMR approach for the detection of ligand binding sites on proteins using soluble paramagnetic nitroxide molecules. The method can also be used to determine the electrostatic potential around the protein surface and it changes upon ligand binding.

Selected Publications:

  • Penk A, Danielsson A, Gaardløs M, Montag C, Schöler A, Huster D, Samsonov SA, Künze G. Detecting Protein-Ligand Interactions with Nitroxide Based Paramagnetic Cosolutes. Chemistry. 2024. 30(18):e202303570. doi: 10.1002/chem.202303570
  • Koehler Leman J, Künze G. Recent Advances in NMR Protein Structure Prediction with ROSETTA. Int J Mol Sci. 2023. 24(9):7835. doi: 10.3390/ijms24097835
  • Ledwitch KV, Künze G, McKinney JR, Okwei E, Larochelle K, Pankewitz L, Ganguly S, Darling HL, Coin I, Meiler J. Sparse pseudocontact shift NMR data obtained from a non-canonical amino acid-linked lanthanide tag improves integral membrane protein structure prediction. J Biomol NMR. 2023. 77(3):69-82. doi: 10.1007/s10858-023-00412-9
  • Zehnder J, Cadalbert R, Yulikov M, Künze G, Wiegand T. Paramagnetic spin labeling of a bacterial DnaB helicase for solid-state NMR. J Magn Reson. 2021. 332:107075. doi: 10.1016/j.jmr.2021.107075
  • Kuenze G, Bonneau R, Leman JK, Meiler J. Integrative Protein Modeling in RosettaNMR from Sparse Paramagnetic Restraints. Structure. 2019. 27(11):1721-1734.e5. doi: 10.1016/j.str.2019.08.012
  • Köhling S, Künze G, Lemmnitzer K, Bermudez M, Wolber G, Schiller J, Huster D, Rademann J. Chemoenzymatic Synthesis of Nonasulfated Tetrahyaluronan with a Paramagnetic Tag for Studying Its Complex with Interleukin-10. Chemistry. 2016. 22(16):5563-74. doi: 10.1002/chem.201504459
  • Kuenze G, Köhling S, Vogel A, Rademann J, Huster D. Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy. J Biol Chem. 2016. 291(6):3100-13. doi: 10.1074/jbc.M115.681759

Collaborations:

Prof. Dr. Daniel Huster, Institute for Medical Physics and Biophysics, University of Leipzig

Prof. Dr. Thomas Wiegand, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University

Prof. Dr. Björn Corzilius, Institute of Chemistry, Faculty of Mathematics and Natural Sciences, University of Rostock