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Binding Moiety Mapping by Saturation Transfer Difference NMR

  • Jeffrey R. Brender
  • Janarthanan Krishnamoorthy
  • Anirban Ghosh
  • Anirban Bhunia
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1824)

Abstract

Saturation transfer difference (STD) NMR has emerged as one of the key technologies in lead optimization during drug design. Unlike most biophysical assays which report only on the binding affinity, STD NMR reports simultaneously on both the binding affinity and the structure of the binding ligand/protein complex. The STD experiment drives magnetization from a protein to a bound small molecule ligand which carries away the memory of the saturation signal when it dissociates. Since the transfer of saturation is distance dependent, STD NMR can be used to map the specific atoms on the ligand in contact with a protein receptor allowing the impact of any structural change in the binding site to be mapped directly on to the individual functional groups responsible when a suitable compound library is screened. Because the signal is detected from the free ligand and not the bound complex, it can be used on a much wider range of systems than protein-detected NMR and has the advantage of more directly reporting on distances than changes in chemical shifts alone. The STD experiment, while deceptively simple, is very sensitive to both sample conditions and acquisition parameters. We present a general protocol for setting up and STD NMR experiment with a particular focus on how choices in sample conditions and acquisition parameters affect the outcome of the experiment.

Key words

Nuclear magnetic resonance spectroscopy Saturation transfer difference NMR Epitope mapping Screening Ligand-based NMR 

References

  1. 1.
    Meyer B, Peters T (2003) NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors. Angew Chem Int Ed Eng 42(8):864–890. https://doi.org/10.1002/anie.200390233 CrossRefGoogle Scholar
  2. 2.
    Groftehauge MK, Hajizadeh NR, Swann MJ, Pohl E (2015) Protein-ligand interactions investigated by thermal shift assays (TSA) and dual polarization interferometry (DPI). Acta Crystallogr D Biol Crystallogr 71(Pt 1):36–44. https://doi.org/10.1107/S1399004714016617 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Jerabek-Willemsen M, André T et al (2014) MicroScale Thermophoresis: interaction analysis and beyond. J Mol Struct 1077(Supplement C):101–113. https://doi.org/10.1016/j.molstruc.2014.03.009 CrossRefGoogle Scholar
  4. 4.
    Carpenter JW, Laethem C, Hubbard FR et al (2002) Configuring radioligand receptor binding assays for HTS using scintillation proximity assay technology. Methods Mol Biol 190:31–49. https://doi.org/10.1385/1-59259-180-9:031 CrossRefPubMedGoogle Scholar
  5. 5.
    Patching SG (2014) Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery. Biochim Biophys Acta 1838(1 Pt A):43–55. https://doi.org/10.1016/j.bbamem.2013.04.028 CrossRefPubMedGoogle Scholar
  6. 6.
    Shuker SB, Hajduk PJ, Meadows RP, Fesik SW (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274(5292):1531–1534CrossRefGoogle Scholar
  7. 7.
    Stockman BJ, Dalvit C (2002) NMR screening techniques in drug discovery and drug design. Prog Nucl Magn Reson Spectrosc 41(3–4):187–231. https://doi.org/10.1016/S0079-6565(02)00049-3 CrossRefGoogle Scholar
  8. 8.
    Pellecchia M, Bertini I, Cowburn D et al (2008) Perspectives on NMR in drug discovery: a technique comes of age. Nat Rev Drug Discov 7(9):738–745. https://doi.org/10.1038/nrd2606 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Jameson CJ (1996) Understanding NMR chemical shifts. Annu Rev Phys Chem 47:135–169. https://doi.org/10.1146/annurev.physchem.47.1.135 CrossRefGoogle Scholar
  10. 10.
    de Dios AC, Jameson CJ (2012) Recent advances in nuclear shielding calculations. Annu Rep Nmr Spectro 77:1–80. https://doi.org/10.1016/B978-0-12-397020-6.00001-5 CrossRefGoogle Scholar
  11. 11.
    Anglister J, Srivastava G, Naider F (2016) Detection of intermolecular NOE interactions in large protein complexes. Prog Nucl Magn Reson Spectrosc 97:40–56. https://doi.org/10.1016/j.pnmrs.2016.08.002 CrossRefPubMedGoogle Scholar
  12. 12.
    Post CB (2003) Exchange-transferred NOE spectroscopy and bound ligand structure determination. Curr Opin Struct Biol 13(5):581–588. https://doi.org/10.1016/j.sbi.2003.09.012 CrossRefPubMedGoogle Scholar
  13. 13.
    Mayer M, Meyer B (1999) Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Ed 38(12):1784–1788. https://doi.org/10.1002/(Sici)1521-3773(19990614)38:12<1784::Aid-Anie1784>3.0.Co;2-QCrossRefGoogle Scholar
  14. 14.
    Mayer M, Meyer B (2001) Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J Am Chem Soc 123(25):6108–6117. https://doi.org/10.1021/ja0100120 CrossRefPubMedGoogle Scholar
  15. 15.
    Bhunia A, Bhattacharjya S, Chatterjee S (2012) Applications of saturation transfer difference NMR in biological systems. Drug Discov Today 17(9–10):505–513. https://doi.org/10.1016/j.drudis.2011.12.016 CrossRefPubMedGoogle Scholar
  16. 16.
    Haselhorst T, Lamerz AC, Itzstein M (2009) Saturation transfer difference NMR spectroscopy as a technique to investigate protein-carbohydrate interactions in solution. Methods Mol Biol 534:375–386. https://doi.org/10.1007/978-1-59745-022-5_26 CrossRefPubMedGoogle Scholar
  17. 17.
    Wagstaff JL, Taylor SL, Howard MJ (2013) Recent developments and applications of saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy. Mol BioSyst 9(4):571–577. https://doi.org/10.1039/c2mb25395j CrossRefPubMedGoogle Scholar
  18. 18.
    Venkitakrishnan RP, Benard O, Max M et al (2012) Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins. Methods Mol Biol 914:47–63. https://doi.org/10.1007/978-1-62703-023-6_4 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Benie AJ, Moser R, Bauml E et al (2003) Virus-ligand interactions: identification and characterization of ligand binding by NMR spectroscopy. J Am Chem Soc 125(1):14–15. https://doi.org/10.1021/ja027691e CrossRefPubMedGoogle Scholar
  20. 20.
    Harris KA, Shekhtman A, Agris PF (2013) Specific RNA-protein interactions detected with saturation transfer difference NMR. RNA Biol 10(8):1307–1311. https://doi.org/10.4161/rna.25948 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Di Micco S, Bassarello C, Bifulco G et al (2006) Differential-frequency saturation transfer difference NMR spectroscopy allows the detection of different ligand-DNA binding modes. Angew Chem Int Ed 45(2):224–228. https://doi.org/10.1002/anie.200501344 CrossRefGoogle Scholar
  22. 22.
    Hens Z, Martins JC (2013) A solution NMR toolbox for characterizing the surface chemistry of colloidal nanocrystals. Chem Mater 25(8):1211–1221. https://doi.org/10.1021/cm303361s CrossRefGoogle Scholar
  23. 23.
    Claasen B, Axmann M, Meinecke R, Meyer B (2005) Direct observation of ligand binding to membrane proteins in living cells by a saturation transfer double difference (STDD) NMR spectroscopy method shows a significantly higher affinity of integrin alpha(IIb)beta3 in native platelets than in liposomes. J Am Chem Soc 127(3):916–919. https://doi.org/10.1021/ja044434w CrossRefPubMedGoogle Scholar
  24. 24.
    Dias DM, Ciulli A (2014) NMR approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes. Prog Biophys Mol Biol 116(2–3):101–112. https://doi.org/10.1016/j.pbiomolbio.2014.08.012 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Ma R, Wang P, Wu J, Ruan K (2016) Process of fragment-based lead discovery-a perspective from NMR. Molecules 21(7). https://doi.org/10.3390/molecules21070854 CrossRefGoogle Scholar
  26. 26.
    Cala O, Krimm I (2015) Ligand-orientation based fragment selection in STD NMR screening. J Med Chem 58(21):8739–8742. https://doi.org/10.1021/acs.jmedchem.5b01114 CrossRefGoogle Scholar
  27. 27.
    Kim HY, Wyss DF (2015) NMR screening in fragment-based drug design: a practical guide. Methods Mol Biol 1263:197–208. https://doi.org/10.1007/978-1-4939-2269-7_16 CrossRefPubMedGoogle Scholar
  28. 28.
    Vanwetswinkel S, Heetebrij RJ, van Duynhoven J et al (2005) TINS, target immobilized NMR screening: an efficient and sensitive method for ligand discovery. Chem Biol 12(2):207–216. https://doi.org/10.1016/j.chembiol.2004.12.004 CrossRefPubMedGoogle Scholar
  29. 29.
    Jayalakshmi V, Krishna NR (2005) Determination of the conformation of trimethoprim in the binding pocket of bovine dihydrofolate reductase from a STD-NMR intensity-restrained CORCEMA-ST optimization. J Am Chem Soc 127(40):14080–14084. https://doi.org/10.1021/ja054192f CrossRefPubMedGoogle Scholar
  30. 30.
    Jayalakshmi V, Biet T, Peters T, Krishna NR (2004) Refinement of the conformation of UDP-galactose bound to galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization. J Am Chem Soc 126(28):8610–8611. https://doi.org/10.1021/ja048703u CrossRefPubMedGoogle Scholar
  31. 31.
    Zhang W, Li R, Shin R, Wang Y et al (2013) Identification of the binding site of an allosteric ligand using STD-NMR, docking, and CORCEMA-ST calculations. ChemMedChem 8(10):1629–1633. https://doi.org/10.1002/cmdc.201300267 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Jayalakshmi V, Krishna NR (2002) Complete relaxation and conformational exchange matrix (CORCEMA) analysis of intermolecular saturation transfer effects in reversibly forming ligand-receptor complexes. J Magn Reson 155(1):106–118. https://doi.org/10.1006/jmre.2001.2499 CrossRefPubMedGoogle Scholar
  33. 33.
    Quiros MT, Macdonald C, Angulo J, Munoz MP (2016) Spin saturation transfer difference NMR (SSTD NMR): a new tool to obtain kinetic parameters of chemical exchange processes. J Vis Exp 117. https://doi.org/10.3791/54499
  34. 34.
    Viegas A, Manso J, Nobrega FL, Cabrita EJ (2011) Saturation-transfer difference (STD) NMR: a simple and fast method for ligand screening and characterization of protein binding. J Chem Educ 88(7):990–994. https://doi.org/10.1021/ed101169t CrossRefGoogle Scholar
  35. 35.
    Kemper S, Patel MK, Errey JC et al (2010) Group epitope mapping considering relaxation of the ligand (GEM-CRL): including longitudinal relaxation rates in the analysis of saturation transfer difference (STD) experiments. J Magn Reson 203(1):1–10. https://doi.org/10.1016/j.jmr.2009.11.015 CrossRefPubMedGoogle Scholar
  36. 36.
    McGovern SL, Caselli E, Grigorieff N, Shoichet BK (2002) A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem 45(8):1712–1722CrossRefPubMedGoogle Scholar
  37. 37.
    Coan KE, Shoichet BK (2008) Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. J Am Chem Soc 130(29):9606–9612. https://doi.org/10.1021/ja802977h CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Aldrich C, Bertozzi C, Georg G et al (2017) The ecstasy and agony of assay interference compounds. J Med Chem 60(6):2165–2168. https://doi.org/10.1021/acs.jmedchem.7b00229 CrossRefPubMedGoogle Scholar
  39. 39.
    Feng BY, Shelat A, Doman TN et al (2005) High-throughput assays for promiscuous inhibitors. Nat Chem Biol 1(3):146–148. https://doi.org/10.1038/nchembio718 CrossRefPubMedGoogle Scholar
  40. 40.
    Feng BY, Simeonov A, Jadhav A et al (2007) A high-throughput screen for aggregation-based inhibition in a large compound library. J Med Chem 50(10):2385–2390. https://doi.org/10.1021/jm061317y CrossRefPubMedGoogle Scholar
  41. 41.
    Harwood JS, Mo H (2016) Practical NMR spectroscopy laboratory guide using Bruker spectrometers. Academic Press, LondonGoogle Scholar
  42. 42.
    Berger S, Braun S (2004) 200 and more NMR experiments: a practical course. 3rd rev. and expanded edn. Wiley, LeipzigGoogle Scholar
  43. 43.
    Hwang TL, Shaka AJ (1995) Water suppression that works. Excitation sculpting using arbitrary wave-forms and pulsed-field gradients. J Magn Reson Ser A 112(2):275–279. https://doi.org/10.1006/jmra.1995.1047 CrossRefGoogle Scholar
  44. 44.
    Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2(6):661–665CrossRefPubMedGoogle Scholar
  45. 45.
    Ley NB, Rowe ML, Williamson RA, Howard MJ (2014) Optimising selective excitation pulses to maximise saturation transfer difference NMR spectroscopy. RSC Adv 4(14):7347–7351. https://doi.org/10.1039/C3RA46246C CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Mitra P, Shultis D, Brender JR et al (2013) An evolution-based approach to De novo protein design and case study on mycobacterium tuberculosis. PLoS Comput Biol 9(10):e1003298. https://doi.org/10.1371/journal.pcbi.1003298 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Bauer C, Freeman R, Frenkiel T et al (1984) Gaussian pulses. J Magn Reson 58(3):442–457. https://doi.org/10.1016/0022-2364(84)90148-3 CrossRefGoogle Scholar
  48. 48.
    Cutting B, Shelke SV, Dragic Z et al (2007) Sensitivity enhancement in saturation transfer difference (STD) experiments through optimized excitation schemes. Magn Reson Chem 45(9):720–724. https://doi.org/10.1002/mrc.2033 CrossRefPubMedGoogle Scholar
  49. 49.
    Claridge TDW, ScienceDirect (Online service) (2009) High-resolution NMR techniques in organic chemistry. Elsevier, AmsterdamGoogle Scholar
  50. 50.
    Yan J, Kline AD, Mo H et al (2003) The effect of relaxation on the epitope mapping by saturation transfer difference NMR. J Magn Reson 163(2):270–276CrossRefPubMedGoogle Scholar
  51. 51.
    Kelly AE, Ou HD, Withers R, Dotsch V (2002) Low-conductivity buffers for high-sensitivity NMR measurements. J Am Chem Soc 124(40):12013–12019CrossRefPubMedGoogle Scholar
  52. 52.
    Voehler MW, Collier G, Young JK et al (2006) Performance of cryogenic probes as a function of ionic strength and sample tube geometry. J Magn Reson 183(1):102–109. https://doi.org/10.1016/j.jmr.2006.08.002 CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Lepre CA, Moore JM, Peng JW (2004) Theory and applications of NMR-based screening in pharmaceutical research. Chem Rev 104(8):3641–3676. https://doi.org/10.1021/cr030409h CrossRefPubMedGoogle Scholar
  54. 54.
    Dalvit C, Flocco M, Knapp S et al (2002) High-throughput NMR-based screening with competition binding experiments. J Am Chem Soc 124(26):7702–7709CrossRefPubMedGoogle Scholar
  55. 55.
    Jahnke W, Floersheim P, Ostermeier C et al (2002) NMR reporter screening for the detection of high-affinity ligands. Angew Chem Int Ed Eng 41(18):3420–3423. https://doi.org/10.1002/1521-3773(20020916)41:18<3420::AID-ANIE3420>3.0.CO;2-ECrossRefGoogle Scholar
  56. 56.
    Siriwardena AH, Tian F, Noble S, Prestegard JH (2002) A straightforward NMR-spectroscopy-based method for rapid library screening. Angew Chem Int Ed Eng 41(18):3454–3457. https://doi.org/10.1002/1521-3773(20020916)41:18<3454::AID-ANIE3454>3.0.CO;2-LCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Jeffrey R. Brender
    • 1
  • Janarthanan Krishnamoorthy
    • 2
  • Anirban Ghosh
    • 3
  • Anirban Bhunia
    • 3
  1. 1.Radiation Biology BranchNational Cancer Institute, National Institutes of HealthBethesdaUSA
  2. 2.Department of BiosciencesJimma UniversityJimmaUSA
  3. 3.Department of BiophysicsBose InstituteKolkataIndia

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