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Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis

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Abstract

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein–ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

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References

  • Anderson MA, Shim H, Raushel FM, Cleland WW (2001) Hydrolysis of phosphotriesters: determination of transition states in parallel reactions by heavy-atom isotope effects. J Am Chem Soc 123:9246–9253

    Article  Google Scholar 

  • Barbieri L, Luchinat E, Banci L (2016) Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells. Nat Protoc 11:1101–1111

    Article  Google Scholar 

  • Bertini I, Luchinat C, Parigi G (2002) Magnetic susceptibility in paramagnetic NMR. Prog Nucl Magn Reson Spectrosc 40:249–273

    Article  Google Scholar 

  • Bertini I, Del Bianco C, Gelis I, Katsaros N, Luchinat C, Parigi G, Peana M, Provenzani A, Zoroddu MA (2004) Experimentally exploring the conformational space sampled by domain reorientation in calmodulin. Proc Natl Acad Sci USA 101:6841–6846

    Article  ADS  Google Scholar 

  • Bertini I, Giachetti A, Luchinat C, Parigi G, Petoukhov MV, Pierattelli R, Ravera E, Svergun DI (2010) Conformational space of flexible biological macromolecules from average data. J Am Chem Soc 132:13553–13558

    Article  Google Scholar 

  • Bertini I, Luchinat C, Nagulapalli M, Parigi G, Ravera E (2012) Paramagnetic relaxation enhancement for the characterization of the conformational heterogeneity in two-domain proteins. Phys Chem Chem Phys 14:9149–9156

    Article  Google Scholar 

  • Camilloni C, Vendruscolo M (2015) Using pseudocontact shifts and residual dipolar couplings as exact NMR restraints for the determination of protein structural ensembles. Biochemistry 54:7470–7476

    Article  Google Scholar 

  • Cao C, Chen JL, Yang Y, Huang F, Otting G, Su XC (2014) Selective 15N-labeling of the side-chain amide groups of asparagine and glutamine for applications in paramagnetic NMR spectroscopy. J Biomol NMR 59:251–261

    Article  Google Scholar 

  • Chen JL, Wang X, Yang F, Cao C, Otting G, Su XC (2016a) 3D structure determination of an unstable transient enzyme intermediate by paramagnetic NMR spectroscopy. Angew Chem Int Ed 55:13744–13748

    Article  Google Scholar 

  • Chen JL, Yang Y, Zhang LL, Liang H, Huber T, Su XC, Otting G (2016b) Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy. Phys Chem Chem Phys 18:5850–5859

    Article  Google Scholar 

  • Clore GM, Iwahara J (2009) Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-population states of biological macromolecules and their complexes. Chem Rev 109:4108–4139

    Article  Google Scholar 

  • Ding Y, Zhao W, Song W, Zhao Z, Ma B (2011) Mild and recyclable catalytic oxidation of pyridines to N-oxides with H2O2 in water mediated by a vanadium-substituted polyoxometalate. Green Chem 13:1486–1489

    Article  Google Scholar 

  • Dosset P, Hus JC, Marion D, Blackledge M (2001) A novel interactive tool for rigid-body modeling of multi-domain macromolecules using residual dipolar couplings. J Biomol NMR 20:223–231

    Article  Google Scholar 

  • Fragai M, Luchinat C, Parigi G, Ravera E (2013) Conformational freedom of metalloproteins revealed by paramagnetism-assisted NMR. Coord Chem Rev 257:2652–2667

    Article  Google Scholar 

  • Gaponenko V, Altieri AS, Li J, Byrd RA (2002) Breaking symmetry in the structure determination of (large) symmetric protein dimers. J Biomol NMR 24:143–148

    Article  Google Scholar 

  • Graham B, Loh CT, Swarbrick JD, Ung P, Shin J, Yagi H, Jia X, Chhabra S, Barlow N, Pintacuda G, Huber T, Otting G (2011) DOTA-amide lanthanide tag for reliable generation of pseudocontact shifts in protein NMR spectra. Bioconjugate Chem 22:2118–2125

    Article  Google Scholar 

  • Guan JY, Keizers PH, Liu WM, Löhr F, Skinner SP, Heeneman EA, Schwalbe H, Ubbink M, Siegal G (2013) Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy. J Am Chem Soc 135:5859–5868

    Article  Google Scholar 

  • Hänsel R, Luh LM, Corbeski I, Trantirek L, Dötsch V (2014) In-cell NMR and EPR spectroscopy of biomacromolecules. Angew Chem Int Ed 53:10300–10314

    Article  Google Scholar 

  • Hass MAS, Ubbink M (2014) Structure determination of protein–protein complexes with long-range anisotropic paramagnetic NMR restraints. Curr Opin Struct Biol 24:45–53

    Article  Google Scholar 

  • Häussinger D, Huang JR, Grzesiek S (2009) DOTA-M8: an extremely rigid, high-affinity lanthanide chelating tag for PCS NMR spectroscopy. J Am Chem Soc 131:14761–14767

    Article  Google Scholar 

  • Hikone Y, Hirai G, Mishima M, Inomata K, Ikeya T, Arai S, Shirakawa M, Sodeoka M, Ito Y (2016) A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells. J Biolmol NMR 66:99–110

    Article  Google Scholar 

  • Ikegami T, Verdier L, Sakhaii P, Grimme S, Pescatore B, Saxena K, Fiebig KM, Griesinger C (2004) Novel techniques for weak alignment of proteins in solution using chemical tags coordinating lanthanide ions. J Biomol NMR 29:339–349

    Article  Google Scholar 

  • Jiang WX, Gu XH, Dong X, Tang C (2017) Lanthanoid tagging via an unnatural amino acid for protein structure characterization. J Biolmol NMR 67:273–282

    Article  Google Scholar 

  • John M, Pintacuda G, Park AY, Dixon NE, Otting G (2006) Structure determination of protein–ligand complexes by transferred paramagnetic shifts. J Am Chem Soc 128:12910–12916

    Article  Google Scholar 

  • Keizers PHJ, Desreux JF, Overhand M, Ubbink M (2007) Increased paramagnetic effect of a lanthanide protein probe by two-point attachment. J Am Chem Soc 29:9292–9293

    Article  Google Scholar 

  • Keizers PHJ, Saragliadis A, Hiruma Y, Overhand M, Ubbink M (2008) 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

    Article  Google Scholar 

  • Kmentova I, Sutherland HS, Palmer BD, Blaser A, Franzblau SG, Wang B, Wang Y, Ma Z, Denny WA, Thompson AM (2010) Synthesis and structure–activity relationships of aza- and diazabiphenyl analogues of the antitubercular drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine (PA-824). J Med Chem 53:8421–8439

    Article  Google Scholar 

  • Koehler J, Meiler J (2011) Expanding the utility of NMR restraints with paramagnetic compounds: background and practical aspects. Prog Nucl Magn Reson Spectrosc 59:360–389

    Article  Google Scholar 

  • Lange OF, Lakomek NA, Farès C, Schröder GF, Walter KFA, Becker S, Meiler J, Grubmüller H, Griesinger C, de Groot BL (2008) Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science 320:1471–1475

    Article  ADS  Google Scholar 

  • Lee MD, Loh CT, Shin J, Chhabra S, Dennis ML, Otting G, Swarbrick JD, Graham B (2015) Compact, hydrophilic, lanthanide-binding tags for paramagnetic NMR spectroscopy. Chem Sci 6:2614–2624

    Article  Google Scholar 

  • Leonov A, Voigt B, Rodriguez-Castañeda F, Sakhaii P, Griesinger C (2005) Convenient synthesis of multifunctional EDTA-based chiral metal chelates substituted with an S-mesylcysteine. Chem Eur J 11:3342–3348

    Article  Google Scholar 

  • Li C, Liu M (2012) Protein dynamics in living cells studied by in-cell NMR spectroscopy. FEBS Lett 587:1008–1011

    Article  Google Scholar 

  • Li QF, Yang Y, Maleckis A, Otting G, Su XC (2012) Thiol–ene reaction: a versatile tool in site-specific labelling of proteins with chemically inert tags for paramagnetic NMR. Chem Commun 48:2704–2706

    Article  Google Scholar 

  • Liu WM, Keizers PHJ, Hass MAS, Blok A, Timmer M, Sarris AJC, Overhand M, Ubbink M (2012) A pH sensitive, colorful, lanthanide-chelating paramagnetic NMR probe. J Am Chem Soc 134:17306–17313

    Article  Google Scholar 

  • Liu WM, Overhand M, Ubbink M (2014a) The application of paramagnetic lanthanoid ions in NMR spectroscopy on proteins. Coord Chem Rev 273:2–12

    Article  Google Scholar 

  • Liu WM, Skinner SP, Timmer M, Blok A, Hass MAS, Filippov DV, Overhand M, Ubbink M (2014b) A two-armed lanthanoid-chelating paramagnetic NMR probe linked to proteins via thioether linkages. Chem Eur J 20:6256–6258

    Article  Google Scholar 

  • Loh CT, Ozawa K, Tuck KL, Barlow N, Huber T, Otting G, Graham B (2013) Lanthanide tags for site-specific ligation to an unnatural amino acid and generation of pseudocontact shifts in proteins. Bioconjug Chem 24:260–268

    Article  Google Scholar 

  • Loh CT, Graham B, Abdelkader EH, Tuck KL, Otting G (2015) Generation of pseudocontact shifts in proteins with lanthanides using small “clickable” nitrilotriacetic acid and iminodiacetic acid tags. Chem Eur J 21:5084–5092

    Article  Google Scholar 

  • Luchinat C (2016) Exploring the conformational heterogeneity of biomolecules: theory and experiments. Phys Chem Chem Phys 18:5684–5685

    Article  Google Scholar 

  • Luchinat E, Banci L (2016) A unique tool for cellular structural biology: in-cell NMR. J Biol Chem 291:3776–3784

    Article  Google Scholar 

  • Luchinat E, Banci L (2017) In-cell NMR: a topical review. IUCrJ 4:108–118

    Article  Google Scholar 

  • Ma FH, Chen JL, Li QF, Zuo HH, Huang F, Su XC (2014) Kinetic assay of the michael addition-like thiol–ene reaction and Insight into protein bioconjugation. Chem Asian J 9:1808–1816

    Article  Google Scholar 

  • Maltsev A, Grishaev A, Roche J, Zasloff M, Bax A (2014) Improved cross validation of a static ubiquitin structure derived from high precision residual dipolar couplings measured in a drug based liquid crystalline phase. J Am Chem Soc 136:3752–3755

    Article  Google Scholar 

  • Marley J, Lu M, Bracken C (2001) A method for efficient isotopic labeling of recombinant proteins. J Biomol NMR 20:71–75

    Article  Google Scholar 

  • Martorana A, Bellapadrona G, Feintuch A, Gregorio ED, Aime S, Goldfarb D (2014) Probing protein conformation in cells by EPR distance measurements using Gd3+ spin labeling. J Am Chem Soc 136:13458–13465

    Article  Google Scholar 

  • Martorana A, Yang Y, Zhao Y, Li QF, Su XC, Goldfarb D (2015) Mn(II) tags for DEER distance measurements in proteins via C–S attachment. Dalton Trans 44:20812–20816

    Article  Google Scholar 

  • Miranda C, Escartí F, Lamarque L, Yunta MJ, Navarro P, García-España E, Jimeno ML (2004) New 1 H-pyrazole-containing polyamine receptors able to complex l-glutamate in water at physiological pH values. J Am Chem Soc 126:823–833

    Article  Google Scholar 

  • Müntener T, Häussinger D, Selenko P, Theillet FX (2016) In-cell protein structures from 2D NMR experiments. J Phys Chem Lett 7:2821–2825

    Article  Google Scholar 

  • Nitsche C, Otting G (2017a) Pseudocontact shifts in biomolecular NMR using paramagnetic metal tags. Prog Nucl Magn Reson Spectrosc 98:20–49

    Article  Google Scholar 

  • Nitsche C, Mahawaththa MC, Becker W, Huber T, Otting G (2017b) Site-selective tagging of proteins by pnictogen-mediated self-assembly. Chem Commun 53:10894–10897

    Article  Google Scholar 

  • Ochiai E (1953) Recent Japanese work on the chemistry of pyridine 1-oxide and related compounds. J Org Chem 18:534–551

    Article  Google Scholar 

  • Ottiger M, Delaglio F, Bax A (1998) Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. J Magn Reson 131:373–378

    Article  ADS  Google Scholar 

  • Otting G (2010) Protein NMR using paramagnetic ions. Annu Rev Biophys 39:387–405

    Article  Google Scholar 

  • Pan BB, Yang F, Ye Y, Wu Q, Li C, Huber T, Su XC (2016) 3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy. Chem Commun 52:10237–10240

    Article  Google Scholar 

  • Pellegatti L, Zhang J, Drahos B, Villette S, Suzenet F, Guillaumet G, Petoud S, Tóth É (2008) Pyridine-based lanthanide complexes: towards bimodal agents operating as near infrared luminescent and MRI reporters. Chem Commun 28:6591–6593

    Article  Google Scholar 

  • Perica T, Chothia C (2010) Ubiquitin—molecular mechanisms for recognition of different structures. Curr Opin Struct Biol 20:367–376

    Article  Google Scholar 

  • Peters F, Maestre-Martinez M, Leonov A, Kovačič L, Becker S, Boelens R, Griesinger C (2011) Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR. J Biomol NMR 51:329–337

    Article  Google Scholar 

  • Pettit LD, Powell KJ (1999) IUPAC stability constants database, version 4.11. Academic Software, Yorks

    Google Scholar 

  • Pintacuda G, Park AY, Keniry MA, Dixon NE, Otting G (2006) Lanthanide labeling offers fast NMR approach to 3D structure determinations of protein-protein complexes. J Am Chem Soc 128:3696–3702

    Article  Google Scholar 

  • Pintacuda G, John M, Su XC, Otting G (2007) NMR structure determination of protein-ligand complexes by lanthanide labeling. Acc Chem Res 40:206–212

    Article  Google Scholar 

  • Plitzko JM, Schuler B, Selenko P (2017) Structural biology outside the box-inside the cell. Curr Opin Struct Biol 46:110–121

    Article  Google Scholar 

  • Prudêncio M, Rohovec J, Peters JA, Tocheva E, Boulanger MJ, Murphy ME, Hupkes HJ, Kosters W, Impagliazzo A, Ubbink M (2004) A caged lanthanide complex as a paramagnetic shift agent for protein NMR. Chem Eur J 10:3252–3260

    Article  Google Scholar 

  • Russo L, Maestre-Martinez M, Wolff S, Becker S, Griesinger C (2013) Interdomain dynamics explored by paramagnetic NMR. J Am Chem Soc 135:17111–17120

    Article  Google Scholar 

  • Saio T, Ogura K, Shimizu K, Yokochi M, Jr Burke TR, Inagaki F (2011) An NMR strategy for fragment-based ligand screening utilizing a paramagnetic lanthanide probe. J Biomol NMR 51:395–408

    Article  Google Scholar 

  • Saio T, Ogura K, Kumeta H, Kobashigawa Y, Shimizu K, Yokochi M, Kodama K, Yamaguchi H, Tsujishita H, Inagaki F (2015) Ligand-driven conformational changes of MurD visualized by paramagnetic NMR. Sci Rep 5:16685

    Article  ADS  Google Scholar 

  • Sarkar M, Li C, Pielark GJ (2013) Soft interactions and crowding. Biophys Rev 5:187–194

    Article  Google Scholar 

  • Schmitz C, Stanton-Cook MJ, Su XC, Otting G, Huber T (2008) Numbat: an interactive software tool for fitting ∆χ-tensors to molecular coordinates using pseudocontact shifts. J Biolmol NMR 41:179–189

    Article  Google Scholar 

  • Smith AE, Zhang Z, Pielak GJ, Li C (2015) NMR studies of protein folding and binding in cells and cell-like environments. Curr Opin Struc Biol 30:7–16

    Article  Google Scholar 

  • Su XC, Otting G (2010) Paramagnetic labelling of proteins and oligonucleotides for NMR. J Biomol NMR 46:101–112

    Article  Google Scholar 

  • Tang C, Iwahara J, Clore GM (2006) Visualization of transient encounter complexes in protein–protein association. Nature 444:383–386

    Article  ADS  Google Scholar 

  • Tang C, Schwieters CD, Clore GM (2007) Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 449:1078–1082

    Article  ADS  Google Scholar 

  • Wang J, Zhao J, Zhang X, Gao J (2000) Synthesis and structural determination of nine-coordinate K2 [Y(dtpa)(H2O)]†§7H2O. Rare Met 19:241–247

    Google Scholar 

  • Yang Y, Li QF, Cao C, Huang F, Su XC (2013) Site-specific labeling of proteins with a chemically stable, high-affinity tag for protein study. Chem Eur J 19:1097–1103

    Article  Google Scholar 

  • Yang Y, Wang JT, Pei YY, Su XC (2015) Site-specific tagging proteins via a rigid, stable and short thiolether tether for paramagnetic spectroscopic analysis. Chem Commun 51:2824–2827

    Article  Google Scholar 

  • Yang F, Wang X, Pan BB, Su XC (2016) Single-armed phenylsulfonated pyridine derivative of DOTA is rigid and stable paramagnetic tag in protein analysis. Chem Commun 52:11535–11538

    Article  Google Scholar 

  • Yang Y, Yang F, Gong YJ, Chen JL, Goldfarb D, Su XC (2017) A reactive, rigid Gd(III) labeling tag for in-cell EPR distance measurements in proteins. Angew Chem Int Ed 56:2914–2918

    Article  Google Scholar 

  • Ye Y, Liu X, Xu G, Liu M, Li C (2015) Direct observation of Ca2+-induced calmodulin conformational transitions in Intact Xenopus laevis oocytes by 19F NMR spectroscopy. Angew Chem Int Ed 54:5328–5330

    Article  Google Scholar 

  • Yuan Y, Guo S (2011) A mild and efficient synthesis of aryl sulfones from aryl chlorides and sulfinic acid salts using microwave heating. Synlett 18:2750–2756

    Google Scholar 

  • Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV (2004) Crystal structures of Staphylococcus aureus sortase A and its substrate complex. J Biol Chem 279:31383–31389

    Article  Google Scholar 

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Acknowledgements

This work was supported by the National Key R&D Program of China (2016YFA0501202) and the Natural Science Foundation of China (21673122 and 21473095).

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    1. State Key Laboratory of Elemento-Organic Chemistry and Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), College of Chemistry, Nankai University, Tianjin, 300071, China

      Jia-Liang Chen, Yu Zhao, Yan-Jun Gong, Bin-Bin Pan, Xiao Wang & Xun-Cheng Su

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    1. Jia-Liang Chen
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    2. Yu Zhao
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    Correspondence to Xun-Cheng Su.

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    Jia-Liang Chen and Yu Zhao have contributed equally.

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    Chen, JL., Zhao, Y., Gong, YJ. et al. Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis. J Biomol NMR 70, 77–92 (2018). https://doi.org/10.1007/s10858-017-0160-3

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