Articles | Volume 3, issue 2
https://doi.org/10.5194/mr-3-169-2022
© Author(s) 2022. 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-3-169-2022
© Author(s) 2022. This work is distributed under
the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
Research article
| 
13 Sep 2022
Research article |
| 13 Sep 2022
| 13 Sep 2022
Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine
Sreelakshmi Mekkattu Tharayil
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Mithun C. Mahawaththa
ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Akiva Feintuch
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
Ansis Maleckis
Latvian Institute of Organic Synthesis, Aizkraukles 21, 1006 Riga, Latvia
Sven Ullrich
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Richard Morewood
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Michael J. Maxwell
ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Thomas Huber
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Christoph Nitsche
Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
Daniella Goldfarb
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel
Gottfried Otting
CORRESPONDING AUTHOR
ARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, Australia
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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.
Damian Van Raad, Gottfried Otting, and Thomas Huber
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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.
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.
Thorsten Bahrenberg, Samuel M. Jahn, Akiva Feintuch, Stefan Stoll, and Daniella Goldfarb
Magn. Reson., 2, 161–173, https://doi.org/10.5194/mr-2-161-2021, https://doi.org/10.5194/mr-2-161-2021, 2021
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Double electron–electron resonance (DEER) provides information on the structure of proteins by attaching two spin labels to the protein at a well-defined location and measuring the distance between them. The sensitivity of the method in terms of the amount of the protein that is needed for the experiment depends strongly on the relaxation properties of the spin label and the composition of the solvent. We show how to set up the experiment for best sensitivity when the solvent is water (H2O).
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.
Hassane EL Mkami, Robert I. Hunter, Paul A. S. Cruickshank, Michael J. Taylor, Janet E. Lovett, Akiva Feintuch, Mian Qi, Adelheid Godt, and Graham M. Smith
Magn. Reson., 1, 301–313, https://doi.org/10.5194/mr-1-301-2020, https://doi.org/10.5194/mr-1-301-2020, 2020
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Through a series of DEER measurements on two Gd rulers, with Gd–Gd distances of 2.1 and 6.0 nm, we show that artefacts commonly observed when measuring short distances can be eliminated by avoiding excitation of the central transition by both the pump and observer pulses. By using a wideband induction mode sample holder at 94 GHz, we demonstrate that high-quality DEER measurements will become possible using Gd spin labels at sub-µM concentrations, with implications for in-cell DEER measurements.
Marie Ramirez Cohen, Akiva Feintuch, Daniella Goldfarb, and Shimon Vega
Magn. Reson., 1, 45–57, https://doi.org/10.5194/mr-1-45-2020, https://doi.org/10.5194/mr-1-45-2020, 2020
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DNP is a method used to enhance NMR signals by transferring polarization from radicals to nuclear spins using microwave irradiation. The EPR spectrum of the radical during the irradiation is determined by a process called spectral diffusion and affects the DNP efficiency. This process can be measured by electron–electron double resonance. We explored experimentally and theoretically the contribution of the hyperfine coupling of the 14N nuclei in the nitroxide radical to the spectral diffusion.
Henry William Orton, Thomas Huber, and Gottfried Otting
Magn. Reson., 1, 1–12, https://doi.org/10.5194/mr-1-1-2020, https://doi.org/10.5194/mr-1-1-2020, 2020
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Nuclear magnetic resonance is a technique that allows the measurement of the nanoscale distances between the atoms of a molecule. It is often relied upon for finding the structure of a molecule and how atoms are bonded, but measurements become inaccurate for large distances and therefore for large biological molecules. This research presents a new software for calculating the distances between nuclei and unpaired electrons which offers more accurate long-range distances in biological molecules.
Related subject area
Field: Liquid-state NMR | Topic: Applications – biological macromolecules
NMR side-chain assignments of the Crimean–Congo hemorrhagic fever virus glycoprotein n cytosolic domain
Facilitating the structural characterisation of non-canonical amino acids in biomolecular NMR
Imatinib disassembles the regulatory core of Abelson kinase by binding to its ATP site and not by binding to its myristoyl pocket
Localising nuclear spins by pseudocontact shifts from a single tagging site
Localising individual atoms of tryptophan side chains in the metallo-β-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites
Fluorine NMR study of proline-rich sequences using fluoroprolines
Analysis of conformational exchange processes using methyl-TROSY-based Hahn echo measurements of quadruple-quantum relaxation
Anomalous amide proton chemical shifts as signatures of hydrogen bonding to aromatic sidechains
Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino 1H CEST
High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin
Structural polymorphism and substrate promiscuity of a ribosome-associated molecular chaperone
Small-molecule inhibitors of the PDZ domain of Dishevelled proteins interrupt Wnt signalling
Real-time nuclear magnetic resonance spectroscopy in the study of biomolecular kinetics and dynamics
The long-standing relationship between paramagnetic NMR and iron–sulfur proteins: the mitoNEET example. An old method for new stories or the other way around?
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Fragile protein folds: sequence and environmental factors affecting the equilibrium of two interconverting, stably folded protein conformations
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We used NMR to sequentially assign the side-chain resonances of the cytosolic domain of glycoprotein n of the Crimean–Congo hemorrhagic fever virus. The combination of cell-free protein synthesis with high-field NMR and artificial intelligence approaches facilitated a time- and effort-efficient approach. Our results will be harnessed to study the membrane-bound form of the domain and its interactions with virulence factors, which will ultimately help to understand their role in disease.
Sarah Kuschert, Martin Stroet, Yanni Ka-Yan Chin, Anne Claire Conibear, Xinying Jia, Thomas Lee, Christian Reinhard Otto Bartling, Kristian Strømgaard, Peter Güntert, Karl Johan Rosengren, Alan Edward Mark, and Mehdi Mobli
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Stephan Grzesiek, Johannes Paladini, Judith Habazettl, and Rajesh Sonti
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We show here that binding of the anticancer drug imatinib to the ATP site of Abelson kinase and not binding to its allosteric site coincides with the opening of the kinase regulatory core at nanomolar concentrations. This has implications for the understanding of Abelson’s kinase regulation and activity during medication as well as for the design of new Abelson kinase inhibitors.
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Christopher A. Waudby and John Christodoulou
Magn. Reson., 2, 777–793, https://doi.org/10.5194/mr-2-777-2021, https://doi.org/10.5194/mr-2-777-2021, 2021
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Bei Liu, Atul Rangadurai, Honglue Shi, and Hashim M. Al-Hashimi
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Chih-Ting Huang, Yei-Chen Lai, Szu-Yun Chen, Meng-Ru Ho, Yun-Wei Chiang, and Shang-Te Danny Hsu
Magn. Reson., 2, 375–386, https://doi.org/10.5194/mr-2-375-2021, https://doi.org/10.5194/mr-2-375-2021, 2021
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Nestor Kamdem, Yvette Roske, Dmytro Kovalskyy, Maxim O. Platonov, Oleksii Balinskyi, Annika Kreuchwig, Jörn Saupe, Liang Fang, Anne Diehl, Peter Schmieder, Gerd Krause, Jörg Rademann, Udo Heinemann, Walter Birchmeier, and Hartmut Oschkinat
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György Pintér, Katharina F. Hohmann, J. Tassilo Grün, Julia Wirmer-Bartoschek, Clemens Glaubitz, Boris Fürtig, and Harald Schwalbe
Magn. Reson., 2, 291–320, https://doi.org/10.5194/mr-2-291-2021, https://doi.org/10.5194/mr-2-291-2021, 2021
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The folding, refolding and misfolding of biomacromolecules including proteins, DNA and RNA is an important area of biophysical research to understand functional and disease states of a cell. NMR spectroscopy provides detailed insight, with both high time and atomic resolution. These experiments put stringent requirements on signal-to-noise for often irreversible folding reactions. The review describes methodological approaches and highlights key applications.
Francesca Camponeschi, Angelo Gallo, Mario Piccioli, and Lucia Banci
Magn. Reson., 2, 203–221, https://doi.org/10.5194/mr-2-203-2021, https://doi.org/10.5194/mr-2-203-2021, 2021
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The iron–sulfur cluster binding properties of human mitoNEET have been investigated by 1D and 2D 1H paramagnetic NMR spectroscopy. The NMR spectra of both oxidized and reduced mitoNEET are significantly different from those reported previously for other [Fe2S2] proteins. Our findings revealed the unique electronic properties of mitoNEET and suggests that the specific electronic structure of the cluster possibly drives the functional properties of different [Fe2S2] proteins.
Mitsuhiro Takeda, Yohei Miyanoiri, Tsutomu Terauchi, and Masatsune Kainosho
Magn. Reson., 2, 223–237, https://doi.org/10.5194/mr-2-223-2021, https://doi.org/10.5194/mr-2-223-2021, 2021
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Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the lysine side chain presumably contribute to the structures and functions of proteins, the dual nature of the lysine residue has not been fully understood yet, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain at the atomic scale. We describe herein a novel strategy to address the current situation using nuclear magnetic resonance spectroscopy.
Xingjian Xu, Igor Dikiy, Matthew R. Evans, Leandro P. Marcelino, and Kevin H. Gardner
Magn. Reson., 2, 63–76, https://doi.org/10.5194/mr-2-63-2021, https://doi.org/10.5194/mr-2-63-2021, 2021
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While most proteins adopt one conformation, several interconvert between two or more very different structures. Knowing how sequence changes and small-molecule binding can control this behavior is essential for both understanding biology and inspiring new “molecular switches” which can control cellular pathways. This work contributes by examining these topics in the ARNT protein, showing that features of both the folded and unfolded states contribute to the interconversion process.
Rubin Dasgupta, Karthick B. S. S. Gupta, Huub J. M. de Groot, and Marcellus Ubbink
Magn. Reson., 2, 15–23, https://doi.org/10.5194/mr-2-15-2021, https://doi.org/10.5194/mr-2-15-2021, 2021
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A method is demonstrated that can help in sequence-specific NMR signal assignment to nuclear spins near a strongly paramagnetic metal in an enzyme. A combination of paramagnetically tailored NMR experiments and second-shell mutagenesis was used to attribute previously observed chemical exchange processes in the active site of laccase to specific histidine ligands. The signals of nuclei close to the metal can be used as spies to unravel the role of motions in the catalytic process.
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
Short summary
Short summary
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
Abdelkader, E. H., Feintuch, A., Yao, X., Adams, L. A., Aurelio, L., Graham, B., Goldfarb, D., and Otting, G.:
Protein conformation by EPR spectroscopy using gadolinium tags clicked to genetically encoded p-azido-l-phenylalanine, Chem. Commun., 51, 15898–15901, https://doi.org/10.1039/C5CC07121F, 2015.
Bahrenberg, T., Rosenski, Y., Carmieli, R., Zibzener, K., Qi, M., Frydman, V., Godt, A., Goldfarb, D., and Feintuch, A.:
Improved sensitivity for W-band Gd(III)-Gd(III) and nitroxide-nitroxide DEER measurements with shaped pulses, J. Magn. Reson., 283, 1–13, https://doi.org/10.1016/j.jmr.2017.08.003, 2017.
Barak, N. N., Neumann, P., Sevvana, M., Schutkowski, M., Naumann, K., Malešević, M., Reichardt, H., Fischer, G., Stubbs, M. T., and Ferrari, D. M.:
Crystal structure and functional analysis of the protein disulfide isomerase-related protein ERp29, J. Mol. Biol., 385, 1630–1642, https://doi.org/10.1016/j.jmb.2008.11.052, 2009.
Bertini, I., Luchinat, C., and Parigi, G.:
Magnetic susceptibility in paramagnetic NMR, Prog. Nucl. Mag. Res. Sp., 40, 211–236, https://doi.org/10.1016/S0079-6565(02)00002-X, 2002.
Collauto, A., Frydman, V., Lee, M., Abdelkader, E., Feintuch, A., Swarbrick, J., Graham, B., Otting, G., and Goldfarb, D.:
RIDME distance measurements using Gd(III) tags with a narrow central transition, Phys. Chem. Chem. Phys., 18, 19037–19049, https://doi.org/10.1039/C6CP03299K, 2016.
Corbett, K. D. and Berger, J. M.: Structure of the ATP-binding domain of Plasmodium falciparum Hsp90, Proteins, 78, 2738–2744, https://doi.org/10.1002/prot.22799, 2010.
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.
Garbuio, L., Bordignon, E., Brooks, E. K., Hubbell, W. L., Jeschke, G., and Yulikov, M.:
Orthogonal spin labeling and Gd(III)–nitroxide distance measurements on bacteriophage T4-lysozyme, J. Phys. Chem. B, 117, 3145–3153, https://doi.org/10.1021/jp401806g, 2013.
Giannoulis, A., Ben-Ishay, Y., and Goldfarb, D.:
Characteristics of Gd(III) spin labels for the study of protein conformations, Methods Enzymol., 651, 235–290, https://doi.org/10.1016/bs.mie.2021.01.040, 2021.
Goldfarb, D.: DEER data, Version 2, Zenodo [data set], https://doi.org/10.5281/zenodo.6579184, 2022.
Goldfarb, D., Lipkin, Y., Potapov, A., Gorodetsky, Y., Epel, B., Raitsimring, A. M., Radoul, M., and Kaminker, I.:
HYSCORE and DEER with an upgraded 95 GHz pulse EPR spectrometer, J. Magn. Reson., 194, 8–15, https://doi.org/10.1016/j.jmr.2008.05.019, 2008.
Guignard, L., Ozawa, K., Pursglove, S. E., Otting, G., and Dixon N. E.:
NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach, FEBS Lett., 524, 159–162, https://doi.org/10.1016/S0014-5793(02)03048-X, 2002.
Herath, I. D., Breen, C., Hewitt, S. H., Berki, T. R., Kassir, A. F., Dodson, C., Judd, M., Jabar, S., Cox, N., and Otting, G.:
A chiral lanthanide tag for stable and rigid attachment to single cysteine residues in proteins for NMR, EPR and time-resolved luminescence studies, Chem.-Eur. J., 27, 13009–13023, https://doi.org/10.1002/chem.202101143, 2021.
Jeschke, G., Chechik, V., Ionita, P., Godt, A., Zimmermann, H., Banham, J., Timmel, C., Hilger, D., and Jung, H.:
DeerAnalysis2006 – a comprehensive software package for analyzing pulsed ELDOR data, Appl. Magn. Reson., 30, 473–498, https://doi.org/10.1007/BF03166213, 2006.
Johansen-Leete, J., Ullrich, S., Fry, S. E., Frkic, R., Bedding, M. J., Aggarwal, A., Ashhurst, A. S., Ekanyake, K. B., Mahawaththa, M. C., Sasi, V. M., Passioura, T., Larance, M., Otting, G., Turville, S., Jackson, C. J., Nitsche, C., and Payne R. J.:
Antiviral cyclic peptides targeting the main protease of SARS-CoV-2, Chem. Sci., 13, 3826–3836, https://doi.org/10.1039/D1SC06750H, 2022.
Jones, D. H., Cellitti, S. E., Hao, X., Zhang, Q., Jahnz, M., Summerer, D., Schultz, P. G., Uno, T., and Geierstanger, B. H.:
Site-specific labeling of proteins with NMR-active unnatural amino acids, J. Biomol. NMR, 46, 89–100, https://doi.org/10.1007/s10858-009-9365-4, 2010.
Joss, D. and Häussinger, D.:
Design and applications of lanthanide chelating tags for pseudocontact shift NMR spectroscopy with biomacromolecules, Prog. Nucl. Mag. Res. Sp., 114–115, 284–312, https://doi.org/10.1016/j.pnmrs.2019.08.002, 2019.
Keizers, P. H. J., Saragliadis, A., Hiruma, Y., Overhand, M., and Ubbink, M.:
Design, synthesis, and evaluation of a lanthanide chelating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment, J. Am. Chem. Soc., 130, 14802–14812, https://doi.org/10.1021/ja8054832, 2008.
Klopp, J., Winterhalter, A., Gébleux, R., Scherer-Becker, D., Ostermeier, C., and Gossert, A. D.:
Cost-effective large-scale expression of proteins for NMR studies, J. Biomol. NMR, 71, 247–262, https://doi.org/10.1007/s10858-018-0179-0, 2018.
Liepinsh, E., Baryshev, M., Sharipo, A., Ingelman-Sundberg, M., Otting, G., and Mkrtchian S.:
Thioredoxin-fold as a homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa homodimer, Structure, 9, 457–471, https://doi.org/10.1016/S0969-2126(01)00607-4, 2001.
Mahawaththa, M. C.: NMR dataset for GB1 with DCP tags, Version 1, Zenodo [data set], https://doi.org/10.5281/zenodo.6585976, 2022.
Maxwell, M. J.: DCP NMR files, Version 1, Zenodo [data set], https://doi.org/10.5281/zenodo.7009072, 2022.
Mekkattu Tharayil, S., Mahawaththa, M. C., Loh, C.-T., Adekoya, I., and Otting, G.:
Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy, Magn. Reson., 2, 1–13, https://doi.org/10.5194/mr-2-1-2021, 2021.
Miao, Q., Nitsche, C., Orton, H., Overhand, M., Otting, G., and Ubbink, M.:
Paramagnetic chemical probes for studying biological macromolecules, Chem. Rev., 122, 9571–9642, https://doi.org/10.1021/acs.chemrev.1c00708, 2022.
Morewood, R. and Nitsche, C.:
A biocompatible stapling reaction for in situ generation of constrained peptides, Chem. Sci., 12, 669–674, https://doi.org/10.1039/D0SC05125J, 2021.
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.
Nitsche, C. and Otting, G.:
Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags, Prog. Nucl. Mag. Res. Sp., 98–99, 20–49, https://doi.org/10.1016/j.pnmrs.2016.11.001, 2017.
Nitsche, C., Mahawaththa, M. C., Becker, W., Huber, T., and Otting, G.:
Site-selective tagging of proteins by pnictogen-mediated self-assembly, Chem. Commun., 53, 10894–10897, https://doi.org/10.1039/C7CC06155B, 2017.
Nitsche, C., Onagi, H., Quek, J.-P., Otting, G., Dahai, L., and Huber, T.:
Biocompatible macrocyclization between cysteine and 2-cyanopyridine generates stable peptide inhibitors, Org. Lett., 21, 4709–4712, https://doi.org/10.1021/acs.orglett.9b01545, 2019.
Orton, H. W. and Otting, G.:
Accurate electron–nucleus distances from paramagnetic relaxation enhancements, J. Am. Chem. Soc., 140, 7688–7697, https://doi.org/10.1021/jacs.8b03858, 2018.
Orton, H. W., Huber, T., and Otting, G.:
Paramagpy: software for fitting magnetic susceptibility tensors using paramagnetic effects measured in NMR spectra, Magn. Reson., 1, 1–12, https://doi.org/10.5194/mr-1-1-2020, 2020.
Orton, H. W., Abdelkader, E. H., Topping, L., Butler, S. J., and Otting, G.:
Localising nuclear spins by pseudocontact shifts from a single tagging site, Magn. Reson., 3, 65–76, https://doi.org/10.5194/mr-3-65-2022, 2022.
Ottiger, M., Delaglio, F., and Bax, A.:
Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra, J. Magn. Reson., 131, 373–378, https://doi.org/10.1006/jmre.1998.1361, 1998.
Otting, G.: Protein NMR using paramagnetic ions, Ann. Rev. Biophys., 39, 387–405, https://doi.org/10.1146/annurev.biophys.093008.131321, 2010.
Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T.:
How to measure and predict the molar absorption coefficient of a protein, Protein Sci., 11, 2411–2423, https://doi.org/10.1002/pro.5560041120, 1995.
Pannier, M., Veit, S., Godt, A., Jeschke, G., and Spiess, H. W.:
Dead-time free measurement of dipole-dipole interactions between electron spins, J. Magn. Reson., 142, 331–340, https://doi.org/10.1006/jmre.1999.1944, 2000.
Patil, N. A., Quek, J. P., Schroeder, B., Morewood, R., Rademann, J., Luo, D., and Nitsche, C.:
2-Cyanoisonicotinamide conjugation: a facile approach to generate potent peptide inhibitors of the Zika virus protease, ACS Med. Chem. Lett., 12, 732–737, https://doi.org/10.1021/acsmedchemlett.0c00657, 2021.
Peti, W., Meiler, J., Brüschweiler, R., and Griesinger, C.:
Model-free analysis of protein backbone motion from residual dipolar couplings, J. Am. Chem. Soc., 124, 5822–5833, https://doi.org/10.1021/ja011883c, 2002.
Potapov, A., Yagi, H., Huber, T., Jergic, S., Dixon, N. E., Otting, G., and Goldfarb, D.:
Nanometer scale distance measurements in proteins using Gd3+ spin labelling, J. Am. Chem. Soc., 132, 9040–9048, https://doi.org/10.1021/ja1015662, 2010.
Prokopiou, G., Lee, M. D., Collauto, A., Abdelkader, E. H., Bahrenberg, T., Feintuch, A., Ramirez-Cohen, M., Clayton, J., Swarbrick, J. D., and Graham, B.:
Small Gd(III) tags for Gd(III)–Gd(III) distance measurements in proteins by EPR spectroscopy, Inorg. Chem., 57, 5048–5059, https://doi.org/10.1021/acs.inorgchem.8b00133, 2018.
Qi, R. and Otting, G.:
Mutant T4 DNA polymerase for easy cloning and mutagenesis, PLOS ONE, 14, e0211065, https://doi.org/10.1371/journal.pone.0211065, 2019.
Shah, A., Roux, A., Starck, M., Mosely, J. A., Stevens, M., Norman, D. G., Hunter, R. I., El Mkami, H., Smith, G. M., and Parker, D.:
A gadolinium spin label with both a narrow central transition and short tether for use in double electron electron resonance distance measurements, Inorg. Chem., 58, 3015–3025, https://doi.org/10.1021/acs.inorgchem.8b02892, 2019.
Sharff, A. J., Rodseth, L. E., Spurlino, J. C., and Quiocho, F. A.:
Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis, Biochemistry, 31, 10657–10663, https://doi.org/10.1021/bi00159a003, 1992.
Shishmarev, D. and Otting, G.:
How reliable are pseudocontact shifts induced in proteins and ligands by mobile paramagnetic tags? A modelling study, J. Biomol. NMR, 56, 203–216, https://doi.org/10.1007/s10858-013-9738-6, 2013.
Stanton-Cook, M. J., Su, X.-C., Otting, G., and Huber, T.:
PyParaTools, http://comp-bio.anu.edu.au/mscook/PPT/ (last access: 1 May 2022), 2014.
Su, X.-C. and Chen, J.-L.:
Site-specific tagging of proteins with paramagnetic ions for determination of protein structures in solution and in cells, Accounts Chem. Res., 52, 1675–1686, https://doi.org/10.1021/acs.accounts.9b00132, 2019.
Tolman, J. R., Al-Hashimi, H. M., Kay, L. E., and Prestegard, J. H.:
Structural and dynamic analysis of residual dipolar coupling data for proteins, J. Am. Chem. Soc., 123, 1416–1424, https://doi.org/10.1021/ja002500y, 2001.
Ullrich, S., George, J., Coram, A., Morewood, R., and Nitsche, C.:
Biocompatible and selective generation of bicyclic peptides, Angew. Chem. Int. Ed., https://doi.org/10.1002/ange.202208400, 2022.
Vögeli, B., Yao, L., and Bax, A.:
Protein backbone motions viewed by intraresidue and sequential HN-Ha residual dipolar couplings, J. Biomol. NMR, 41, 17–28, https://doi.org/10.1007/s10858-008-9237-3, 2008.
Welegedara, A. P., Yang, Y., Lee, M. D., Swarbrick, J. D., Huber, T., Graham, B., Goldfarb, D., and Otting, G.:
Double-arm lanthanide tags deliver narrow Gd3+–Gd3+ distance distributions in double electron–electron resonance (DEER) measurements, Chem.-Eur. J., 23, 11694–11702, https://doi.org/10.1039/c5cp02602d, 2017.
Welegedara, A. P., Adams, L. A., Huber, T., Graham, B., and Otting, G.:
Site-specific incorporation of selenocysteine by genetic encoding as a photocaged unnatural amino acid, Bioconjugate Chem., 29, 2257–2264, https://doi.org/10.1021/acs.bioconjchem.8b00254, 2018.
Welegedara, A. P., Maleckis, A., Bandara, R., Mahawaththa, M. C., Herath, I. D., Jiun Tan, Y., Giannoulis, A., Goldfarb, D., Otting, G., and Huber, T.:
Cell-Free synthesis of selenoproteins in high yield and purity for selective protein tagging, ChemBioChem, 22, 1480–1486, https://doi.org/10.1002/cbic.202000785, 2021.
Widder, P., Berner, F., Summerer, D., and Drescher, M.:
Double nitroxide labeling by copper-catalyzed azide–alkyne cycloadditions with noncanonical amino acids for electron paramagnetic resonance spectroscopy, ACS Chem. Biol., 14, 839–844, https://doi.org/10.1021/acschembio.8b01111, 2019.
Worswick, S. G., Spencer, J. A., Jeschke, G., and Kuprov, I.:
Deep neural network processing of DEER data, Sci. Adv., 4, eaat5218, https://doi.org/10.1126/sciadv.aat5218, 2018.
Wu, Z., Lee, M. D., Carruthers, T. J., Szabo, M., Dennis, M. L., Swarbrick, J. D., Graham, B., and Otting, G.:
New lanthanide tag for the generation of pseudocontact shifts in DNA by site-specific ligation to a phosphorothioate group, Bioconjugate Chem., 28, 1741–1748, https://doi.org/10.1021/acs.bioconjchem.7b00202, 2017.
Yagi, H., Banerjee, D., Graham, B., Huber, T., Goldfarb, D., and Otting, G.:
Gadolinium tagging for high-precision measurements of 6 nm distances in protein assemblies by EPR, J. Am. Chem. Soc., 133, 10418–10421, https://doi.org/10.1021/ja204415w, 2011.
Zhang, L., Lin, D., Sun, X., Curth, U., Drosten, C., Sauerhering, L., Becker, S., Rox, K., and Hilgenfeld, R.:
Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors, Science, 368, 409–412, https://doi.org/10.1126/science.abb3405, 2020.
Short summary
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.
Having shown that tagging a protein at a single site with different lanthanoid complexes...
