Articles | Volume 6, issue 2
https://doi.org/10.5194/mr-6-131-2025
© Author(s) 2025. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
https://doi.org/10.5194/mr-6-131-2025
© Author(s) 2025. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Inter-residue through-space scalar 19F–19F couplings between CH2F groups in a protein
Yi Jiun Tan
ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Elwy H. Abdelkader
ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Iresha D. Herath
ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Ansis Maleckis
Latvian Institute of Organic Synthesis, Aizkraukles 21, 1006 Riga, Latvia
Gottfried Otting
CORRESPONDING AUTHOR
ARC Centre of Excellence for Innovations in Peptide and Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
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Elwy H. Abdelkader, Nicholas F. Chilton, Ansis Maleckis, and Gottfried Otting
Magn. Reson. Discuss., https://doi.org/10.5194/mr-2025-12, https://doi.org/10.5194/mr-2025-12, 2025
Preprint under review for MR
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The small protein GB1, where all valine residues were replaced by fluorinated analogues containing one or two CH2F groups, produces 19F NMR spectra with exceptional resolution. We establish a convenient strategy for their assignment and analyse the rotameric states of the CH2F groups by virtue of 3-bond coupling constants and a γ-effect on 13C chemical shifts, which is underpinned by DFT calculations. Transient fluorine-fluorine contacts are documented by through-space 19F-19F couplings.
Damian Van Raad, Gottfried Otting, and Thomas Huber
Magn. Reson., 4, 187–197, https://doi.org/10.5194/mr-4-187-2023, https://doi.org/10.5194/mr-4-187-2023, 2023
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A novel cell-free protein synthesis system called eCells produces amino acids based on specific isotopes using low-cost precursors. The system selectively labels methyl groups, i.e valine and leucine, with high efficiency. eCells achieve high levels of 13C incorporation and deuteration in protein preparations, making them suitable for NMR experiments of large protein complexes. They are easy to prepare, can be scaled up in volume and are a promising tool for protein production and NMR studies.
Sreelakshmi Mekkattu Tharayil, Mithun C. Mahawaththa, Akiva Feintuch, Ansis Maleckis, Sven Ullrich, Richard Morewood, Michael J. Maxwell, Thomas Huber, Christoph Nitsche, Daniella Goldfarb, and Gottfried Otting
Magn. Reson., 3, 169–182, https://doi.org/10.5194/mr-3-169-2022, https://doi.org/10.5194/mr-3-169-2022, 2022
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Having shown that tagging a protein at a single site with different lanthanoid complexes delivers outstanding structural information at a selected site of a protein (such as active sites and ligand binding sites), we now present a simple way by which different lanthanoid complexes can be assembled on a highly solvent-exposed cysteine residue. Furthermore, the chemical assembly is selective for selenocysteine, if a selenocysteine residue can be introduced into the protein of interest.
Henry W. Orton, Elwy H. Abdelkader, Lydia Topping, Stephen J. Butler, and Gottfried Otting
Magn. Reson., 3, 65–76, https://doi.org/10.5194/mr-3-65-2022, https://doi.org/10.5194/mr-3-65-2022, 2022
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Installing a tag containing a paramagnetic metal ion on a protein can lead to large changes (pseudocontact shifts) in the resonances observed in NMR spectra. These are easily measured and contain valuable long-range structural information. The present work shows that a single tagging site furnished with different tags can be sufficient to localise atoms in proteins with high accuracy. In fact, this strategy works almost as well as the same number of tags distributed over multiple tagging sites.
Henry W. Orton, Iresha D. Herath, Ansis Maleckis, Shereen Jabar, Monika Szabo, Bim Graham, Colum Breen, Lydia Topping, Stephen J. Butler, and Gottfried Otting
Magn. Reson., 3, 1–13, https://doi.org/10.5194/mr-3-1-2022, https://doi.org/10.5194/mr-3-1-2022, 2022
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This paper explores a method for determining the solution structure of a solvent-exposed polypeptide segment (the L3 loop), which is next to the active site of the penicillin-degrading enzyme IMP-1. Tagging three different sites on the protein with paramagnetic metal ions allowed positioning of the L3 loop with atomic resolution. It was found that the method was more robust when omitting data obtained with different metal ions if obtained with the same tag at the same tagging site.
Sreelakshmi Mekkattu Tharayil, Mithun Chamikara Mahawaththa, Choy-Theng Loh, Ibidolapo Adekoya, and Gottfried Otting
Magn. Reson., 2, 1–13, https://doi.org/10.5194/mr-2-1-2021, https://doi.org/10.5194/mr-2-1-2021, 2021
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A new way is presented for creating lanthanide binding sites on proteins using site-specifically introduced phosphoserine residues. The paramagnetic effects of lanthanides generate long-range effects, which contain structural information and are readily measured by NMR spectroscopy. Excellent correlations between experimentally observed and back-calculated pseudocontact shifts attest to very good immobilization of the lanthanide ions relative to the proteins.
Cited articles
Alexeev, D., Barlow, P. N., Bury, S. M., Charrier, J.-D., Cooper, A., Hadfield, D., Jamieson, C., Kelly, S. M., Layfield, R., Mayer, R. J., McSparron, H., Price, N. C., Ramage, R., Sawyer, L., Starkmann, B. A., Uhrin, D., Wilken, J., and Young, D. W.: Synthesis, structural and biological studies of ubiquitin mutants containing (2S, 4S)-5-fluoroleucine residues strategically placed in the hydrophobic core, ChemBioChem, 4, 894–896, https://doi.org/10.1002/cbic.200300699, 2003.
Apponyi, M. A., Ozawa, K., Dixon, N. E., and Otting, G.: Cell-free protein synthesis for analysis by NMR spectroscopy, Methods Mol. Biol., 426, 257–268, https://doi.org/10.1007/978-1-60327-058-8_16, 2008.
August, R. A., Khan, J. A., Moody, C. M., and Young, D. W.: Stereospecific synthesis of (2S,4R)-[5,5,5-2H3]leucine, J. Chem. Soc.-Perk. T. 1, 1, 507–514, https://doi.org/10.1039/p19960000507, 1996.
Braunschweiler, L. and Ernst, R. R.: Coherence transfer by isotropic mixing – application to proton correlation spectroscopy, J. Magn. Reson., 53, 512–528, https://doi.org/10.1016/0022-2364(83)90226-3, 1983.
Charrier, J.-D., Hadfield, D. S., Hitchcock, P. B., and Young, D. W.: Synthesis of (2S,4S)- and (2S,4R)-5-fluoroleucine and (2S,4S)-[5,5-2H2]-5-fluoroleucine, Org. Biomol. Chem., 2, 474–482, https://doi.org/10.1039/b314933a, 2004.
Ernst, L. and Ibrom, K.: A new quantitative description of the distance dependence of through-space 19F, 19F spin–spin coupling, Angew. Chem. Int. Edit., 34, 1881–1882, https://doi.org/10.1002/anie.199518811, 1995.
Feeney, J., McCormick, J. E., Bauer, C. J., Birdsall, B., Moody, C. M., Starkmann, B. A., Young, D. W., Francis, P., Havlin, R. H., Arnold, W. D., and Oldfield, E.: 19F nuclear magnetic resonance chemical shifts of fluorine containing aliphatic amino acids in proteins: studies on Lactobacillus casei dihydrofolate reductase containing (2S,4S)-5-fluoroleucine, J. Am. Chem. Soc., 118, 8700–8706, https://doi.org/10.1021/ja960465i, 1996.
Frericks Schmidt, H. L., Sperling, L. J., Gao, Y. G., Wylie, B. J., Boettcher, J. M., Wilson, S. R., and Rienstra, C. M. J.: Crystal polymorphism of protein GB1 examined by solid-state NMR spectroscopy and X-ray diffraction, Phys. Chem. B, 111, 14362–14369, https://doi.org/10.1021/jp075531p, 2007.
Frkic, R. L., Tan, Y. J., Abdelkader, E. H., Maleckis, A., Tarcoveanu, E., Nitsche, C., Otting, G., and Jackson, C. J.: Conformational preferences of the non-canonical amino acids (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, and 5,5'-difluoroleucine in a protein, Biochemistry, 63, 1388–1394, https://doi.org/10.1021/acs.biochem.4c00081, 2024a.
Frkic, R., Tan, Y. J., Maleckis, A., Otting, G., and Jackson, C. J.: 1.3 Å crystal structure of E. coli peptidyl–prolyl isomerase B with uniform substitution of valine by (2S,3S)-4-fluorovaline reveals structure conservation and multiple staggered rotamers of CH2F groups, Biochemistry, 63, 2602–2608, https://doi.org/10.1021/acs.biochem.4c00345, 2024b.
Gallagher, T, Alexander, P., Bryan, P., and Gilliland, G. L.: Two crystal structures of the B1 immunoglobulin-binding domain of streptococcal protein G and comparison with NMR, Biochemistry, 33, 4721–4729, https://doi.org/10.1021/bi00181a032, 1994.
Goehlert, V. A., Krupinska, E., Regan, L., and Stone, M. J.: Analysis of side chain mobility among protein G B1 domain mutants with widely varying stabilities, Protein Sci., 13, 3322–3330, https://doi.org/10.1110/ps.04926604, 2004.
Gopinathan, M. S. and Narasimhan, P. T.: Finite perturbation molecular orbital approach to the dihedral angle dependence of vicinal H-H and H-F couplings, Mol. Phys., 21, 1141–1144, https://doi.org/10.1080/00268977100102271, 1971.
Hierso, J.-C.: Indirect nonbonded nuclear spin–spin coupling: a guide for the recognition and understanding of “through-space” NMR J constants in small organic, organometallic, and coordination compounds, Chem. Rev., 114, 4838–4867, https://doi.org/10.1021/cr400330g, 2014.
Kimber, B. J., Feeney, J., Roberts, G. C. K., Birdsall, B., Griffiths, D. V., Burgen, A. S. V., and Sykes, B. D.: Proximity of two tryptophan residues in dihydrofolate reductase determined by 19F NMR, Nature, 271, 184–185, https://doi.org/10.1038/271184a0, 1978.
Kuszewski, K., Gronenborn, A. M., and Clore, G. M.: Improving the packing and accuracy of NMR structures with a pseudopotential for the radius of gyration, J. Am. Chem. Soc., 121, 2337–2338, https://doi.org/10.1021/ja9843730, 1999.
Lu, T., Zhang, J., Chen, J., Gou, Q., Xia, Z., and Feng, G.: Structure and non-covalent interactions of 1,3-difluoropropane and its complex with water explored by rotational spectroscopy and quantum chemical calculations, J. Chem. Phys., 150, 064305, https://doi.org/10.1063/1.5079564, 2019.
MacKenzie, K. R., Prestegard, J. H., and Engelman, D. M.: Leucine side-chain rotamers in a glycophorin A transmembrane peptide as revealed by three-bon carbon–carbon couplings and 13C chemical shifts, J. Biomol. NMR, 7, 256–260, https://doi.org/10.1007/BF00202043, 1996.
Mallory, F. B., Mallory, C. W., Butler, K. E., Lewis, M. B., Xia, A. Q., Luzik, E. D., Fredenburgh, L. E., Ramanjulu, M. M., Van, Q. N., Francl, M. M., Freed, D. A., Wray, C. C., Hann, C., Nerz-Stormes, M., Carroll, P. J., and Chirlian, L. E.: Nuclear spin–spin coupling via nonbonded interactions. 8. The distance dependence of through-space fluorine–fluorine coupling, J. Am. Chem. Soc., 122, 4108–4116, https://doi.org/10.1021/ja993032z, 2000.
Maleckis, A., Abdelkader, E. H., Herath, I. D., and Otting, G.: Synthesis of fluorinated leucines, valines and alanines for use in protein NMR, Org. Biomol. Chem., 20, 2424–2432, https://doi.org/10.1039/D2OB00145D, 2022.
Marstokk, K.-M. and Møllendal, H.: Structural and conformational properties of 1,3-difluoropropane as studied by microwave spectroscopy and ab initio calculations, Acta Chem. Scand., 51, 1058–1065, https://doi.org/10.3891/acta.chem.scand.51-1058, 1997.
Moody, C. M., Starkmann, B. A., and Young, D. W.: Synthesis of (2S,4S)-5-fluoroleucine, Tetrahedron Lett., 35, 5485–5488, https://doi.org/10.1016/S0040-4039(00)73531-3, 1994.
Neylon, C., Brown, S. E., Kralicek, A. V., Miles, C. S., Love, C. A., and Dixon, N. E.: Interaction of the Escherichia coli replication terminator protein (Tus) with DNA: a model derived from DNA-binding studies of mutant proteins by surface plasmon resonance, Biochemistry, 39, 11989–11999, https://doi.org/10.1021/bi001174w, 2000.
Orton, H. W., Qianzhu, H., Abdelkader, E. H., Tan, Y. J., Habel, E. I., Frkic, R. L., Jackson, C. J., Huber, T., and Otting, G.: Through-space scalar 19F–19F couplings between fluorinated non-canonical amino acids for the detection of specific contacts in proteins, J. Am. Chem. Soc., 143, 19587–19598, https://doi.org/10.1021/jacs.1c10104, 2021.
Otting, G.: NMR spectra of GB1 produced with fluorinated leucine along with spectra of the wild-type reference, Zenodo [data set], https://doi.org/10.5281/zenodo.15266133, 2025.
Otting, G., Liepinsh, E., and Wüthrich, K.: Protein hydration in aqueous solution, Science, 254, 974–980, https://doi.org/10.1126/science.1948083, 1991.
Ozawa, K., Loscha, K. V., Kuppan, K. V., Loh, C. T., Dixon, N. E., and Otting, G.: High yield cell-free protein synthesis for site-specific incorporation of unnatural amino acids at two sites, Biochem. Biophys. Res. Commun., 418, 652–656, https://doi.org/10.1016/j.bbrc.2012.01.069, 2012.
Qianzhu, H., Welegedara, A. P., Williamson, H., McGrath A. E., Mahawaththa, M. C., Dixon, N. E., Otting, G., and Huber, T.: Genetic encoding of para-pentafluorsulfanyl phenylalanine: a highly hydrophobic and strongly electronegative group for stable protein interactions, J. Am. Chem. Soc., 142, 17277–17281, https://doi.org/10.1021/jacs.0c07976, 2020.
Qianzhu, H., Abdelkader, E. H., Herath, I. D., Otting, G., and Huber, T.: Site-specific incorporation of 7-fluoro-L-tryptophan into proteins by genetic encoding to monitor ligand binding by 19F NMR spectroscopy, ACS Sens., 7, 44–49, https://doi.org/10.1021/acssensors.1c02467, 2022.
Qianzhu, H., Abdelkader, E. H., Otting, G., and Huber, T.: Genetic encoding of fluoro-L-tryptophans for site-specific detection of conformational heterogeneity in proteins by NMR spectroscopy, J. Am. Chem. Soc., 146, 13641–13650, https://doi.org/10.1021/jacs.4c03743, 2024.
Sharaf, N. G., and Gronenborn, A. M.: 19F-modified proteins and 19F-containing ligands as tools in solution NMR studies of protein interactions, Methods Enzymol., 565, 67–95, https://doi.org/10.1016/bs.mie.2015.05.014, 2015.
Tan, Y. J., Abdelkader, E. H., Tarcoveanu, E., Maleckis, A., Nitsche, C., and Otting, G.: (2S,4S)-5-Fluoroleucine, (2S,4R)-5-fluoroleucine, and 5,5'-difluoroleucine in E. coli PpiB: protein production, 19F NMR, and ligand sensing enhanced by the γ-gauche effect, Biochemistry, 63, 1376–1387, https://doi.org/10.1021/acs.biochem.4c00080, 2024.
Williamson, K. L., Hus, Y.-F. L., Hall, F. H., Swager, S., and Coulter, M. S.: Dihedral angle and bond angle dependence of vicinal proton–fluorine spin–spin coupling, J. Am. Chem. Soc., 90, 6717–6722, https://doi.org/10.1021/ja01026a028, 1968.
Wu, D., Tian, A., and Sun, H.: Conformational properties of 1,3-difluoropropane, J. Phys. Chem. A, 102, 9901–9905, https://doi.org/10.1021/jp982164w, 1998.
Wu, P. S. C., Ozawa, K., Lim, S. P., Vasudevan, S. G., Dixon N. E., and Otting, G.: Cell-free transcription/translation from PCR-amplified DNA for high-throughput NMR studies, Angew. Chem. Int. Edit., 46, 3356–3358, https://doi.org/10.1002/anie.200605237, 2007.
Short summary
A protein is produced where a single amino acid type is substituted globally by a fluorinated analogue. Through-space fluorine–fluorine contacts are observed by 19F NMR (nuclear magnetic resonance) spectroscopy. Substitution of methyl groups by CH2F groups yields outstanding spectral resolution with minimal structural perturbation of the protein. Our work identifies the γ-gauche effect as the main reason for the spectral dispersion.
A protein is produced where a single amino acid type is substituted globally by a fluorinated...