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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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...
