SH2 Domains pp 269-290 | Cite as

NMR Chemical Shift Mapping of SH2 Peptide Interactions

  • Marissa A. McKercher
  • Deborah S. WuttkeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1555)


Heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiments offer a rapid and high resolution approach to gaining binding and conformational insights into a protein–peptide interaction. By tracking 1H and 15N chemical shift changes over the course of a peptide titration into isotopically labeled protein, amide NH pairs of amino acids whose chemical environment changes upon peptide binding can be identified. When mapped onto a structure of the protein, this approach can identify the peptide-binding interface or regions undergoing conformation changes within a protein upon ligand binding. Monitoring NMR chemical shift changes can also serve as a screening technique to identify novel interaction partners for a protein or to determine the binding affinity of a weak protein–peptide interaction. Here, we describe the application of NMR chemical shift mapping to the study of peptide binding to the C-terminal SH2 domain of PLCγ1.

Key words

Nuclear magnetic resonance (NMR) SH2 domain Peptide Chemical shift mapping (CSM) Phosphotyrosine PLCγ1 



We would like to thank our collaborators Dr. Zhongping Tan and Dr. Xiaoyang Guan for the phosphopeptide synthesis, as well as Dr. Julie Forman-Kay for kindly providing us with a PLCγ1-pET11d construct. We would also like to thank Sabrina Hunt and Kathryn Wall for their generous feedback. This work was supported by NSF grant MCB1121842 (to D.S.W.).


  1. 1.
    Pawson T, Gish GD, Nash P (2001) SH2 domains, interaction modules and cellular wiring. Trends Cell Biol 11:504–511CrossRefPubMedGoogle Scholar
  2. 2.
    Yaffe MB (2002) Phosphotyrosine-binding domains in signal transduction. Nat Rev Mol Cell Biol 3:177–186CrossRefPubMedGoogle Scholar
  3. 3.
    Tinti M, Kiemer L, Costa S et al (2013) The SH2 domain interaction landscape. Cell Rep 3:1293–1305CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yu H (1999) Extending the size limit of protein nuclear magnetic resonance. Proc Natl Acad Sci U S A 96:332–334CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814:942–968CrossRefPubMedGoogle Scholar
  6. 6.
    Fielding L (2007) NMR methods for the determination of protein-ligand dissociation constants. Prog Nucl Magn Reson Spectrosc 51:219–242CrossRefGoogle Scholar
  7. 7.
    Rule GS, Hitchens TK (2006) Two dimensional heteronuclear J-correlated spectroscopy. In: Kaptein R (ed) Fundamentals of protein nmr spectroscopy. Springer, Dordrecht, pp 197–211Google Scholar
  8. 8.
    Berman HM, Westbrook J, Feng Z et al (2000) The protein data bank. Nucleic Acids Res 28:235–242CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Liu, B. (2010) SH2 Domain Structures. Accessed 15 April 2015.
  10. 10.
    Arnold K, Bordoli L, Kopp J et al (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201CrossRefPubMedGoogle Scholar
  11. 11.
    Shuker SB, Hajduk PJ, Meadows RP et al (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274:1531–1534CrossRefPubMedGoogle Scholar
  12. 12.
    Ulrich EL, Akutsu H, Doreleijers JF et al (2008) BioMagResBank. Nucleic Acids Res 36:402–408CrossRefGoogle Scholar
  13. 13.
    Rule GS, Hitchens TK (2006) Resonance assignments: heteronuclear methods. In: Kaptein R (ed) Fundamentals of protein NMR spectroscopy. Springer, Dordrecht, pp 277–312Google Scholar
  14. 14.
    Wittekind M, Mueller L (1993) HNCACB, a high-sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the alpha- and beta-carbon resonances in proteins. J Magn Reson Ser B 101:201–205CrossRefGoogle Scholar
  15. 15.
    Yamazaki T, Lee W, Arrowsmith CH et al (1994) A suite of triple resonance NMR experiments for the backbone assignment of 15N, 13C, 2H labeled proteins with high sensitivity. J Am Chem Soc 116:11655–11666CrossRefGoogle Scholar
  16. 16.
    Grzesiek S, Bax A (1992) Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR. J Am Chem Soc 114:6291–6293CrossRefGoogle Scholar
  17. 17.
    Yamazaki T, Lee W, Revington M et al (1994) An HNCA pulse scheme for the backbone assignment of 15N,13C,2H-labeled proteins: Application to a 37-kDa Trp repressor-DNA complex. J Am Chem Soc 116:6464–6465CrossRefGoogle Scholar
  18. 18.
    Clubb RT, Thanabal V, Wagner G (1992) A constant-time three-dimensional triple-resonance pulse scheme to correlate intraresidue 1HN, 15N, and 13C′ chemical shifts in 15N-13C-labelled proteins. J Magn Reson 97:213–217Google Scholar
  19. 19.
    Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry 29:4659–4667CrossRefPubMedGoogle Scholar
  20. 20.
    Rule GS, Hitchens TK (2006) Exchange processes. In: Kaptein R (ed) Fundamentals of protein NMR spectroscopy. Springer, Dordrecht, pp 403–430Google Scholar
  21. 21.
    Cavanagh J, Fairbrother WJ, Palmer AG III et al (2007) Experimental aspects of NMR spectroscopy. In: Protein NMR spectroscopy, 2nd edn. Elsevier Inc., Burlington, VT, pp 234–235Google Scholar
  22. 22.
    Songyang Z, Shoelson SE, Chaudhuri M et al (1993) SH2 domains recognize specific phosphopeptide sequences. Cell 72:767–776CrossRefPubMedGoogle Scholar
  23. 23.
    Ladbury JE, Lemmon MA, Zhou M et al (1995) Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal. Proc Natl Acad Sci U S A 92:3199–3203CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Lian, L., Roberts, G. C. K. (1993) Effects of chemical exchange on NMR spectra. In: Roberts, G. C. K (ed) NMR of Macromolecules: A Practical Approach. Oxford University Press Inc., New York, pp. 153-182.Google Scholar
  25. 25.
    Kemmer G, Keller S (2010) Nonlinear least-squares data fitting in Excel spreadsheets. Nat Protoc 5:267–281CrossRefPubMedGoogle Scholar
  26. 26.
    Harris RK, Becker ED, Cabral De Menezes SM et al (2001) NMR nomenclature. Nuclear spin properties and conventions for chemical shifts. Pure Appl Chem 73:1795–1818CrossRefGoogle Scholar
  27. 27.
    Harris RK, Becker ED, Cabral De Menezes SM et al (2008) Further conventions for NMR shielding and chemical shifts. Pure Appl Chem 80:59–84CrossRefGoogle Scholar
  28. 28.
    Wishart DS, Bigam CG, Yao J et al (1995) 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR 6:135–140CrossRefPubMedGoogle Scholar
  29. 29.
    Delaglio F, Grzesiek S, Vuister GW et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefPubMedGoogle Scholar
  30. 30.
    Vranken WF, Boucher W, Stevens TJ et al (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins Struct Funct Genet 59:687–696CrossRefPubMedGoogle Scholar
  31. 31.
    Schrodinger LLC (2010) The PyMOL Molecular Graphics System, Version Scholar
  32. 32.
    Rule GS, Hitchens TK (2006) Introduction to signal processing. In: Kaptein R (ed) Fundamentals of protein NMR spectroscopy. Springer, Dordrecht, pp 65–88Google Scholar
  33. 33.
    Farmer BT II, Constantine KL, Goldfarb V et al (1996) Localizing the NADP+ binding site on the MurB enzyme by NMR. Nat Struct Biol 3:995–997CrossRefPubMedGoogle Scholar
  34. 34.
    Lennon G, Auffray C, Polymeropoulos M et al (1996) The I.M.A.G.E. consortium: an integrated molecular analysis of genomes and their expression. Genomics 33:151–152CrossRefPubMedGoogle Scholar
  35. 35.
    Herscovitch M, Perkins E, Baltus A et al (2012) Addgene provides an open forum for plasmid sharing. Nat Biotechnol 30:316–317CrossRefPubMedGoogle Scholar
  36. 36.
    Seiler CY, Park JG, Sharma A et al (2014) DNASU plasmid and PSI: biology-materials repositories: resources to accelerate biological research. Nucleic Acids Res 42:1253–1260CrossRefGoogle Scholar
  37. 37.
    Cline J, Braman JC, Hogrefe HH (1996) PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 24:3546–3551CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Teng Q (2013) NMR sample preparation. In: Structural biology: practical NMR applications, 2nd edn. Springer, New York, NY, pp 103–116CrossRefGoogle Scholar
  39. 39.
    Englander SW, Kallenbach NR (1984) Hydrogen exchange and structural dynamics of proteins and nucleic acids. Q Rev Biophys 16:521–655CrossRefGoogle Scholar
  40. 40.
    Kelly AE, Ou HD, Withers R et al (2002) Low-conductivity buffers for high-sensitivity NMR measurements. J Am Chem Soc 124:12013–12019CrossRefPubMedGoogle Scholar
  41. 41.
    Rule GS, Hitchens TK (2006) Practical aspects of N-dimensional data acquisition and processing. In: Kaptein R (ed) Fundamentals of protein NMR spectroscopy. Springer, Dordrecht, pp 313–315Google Scholar
  42. 42.
    Primrose, W. U. (1993) Sample preparation. In: Roberts, G. C. K (ed) NMR of Macromolecules: A Practical Approach. Oxford University Press Inc., New York, pp. 7-33.Google Scholar
  43. 43.
    Schanda P, Brutscher B (2005) Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J Am Chem Soc 127:8014–8015CrossRefPubMedGoogle Scholar
  44. 44.
    Schanda P, Kupce E, Brutscher B (2005) SOFAST-HMQC experiments for recording two-dimensional heteronuclear correlation spectra of proteins within a few seconds. J Biomol NMR 33:199–211CrossRefPubMedGoogle Scholar
  45. 45.
    Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85:2149–2154CrossRefGoogle Scholar
  46. 46.
    Attard TJ, O’Brien-Simpson N, Reynolds EC (2007) Synthesis of phosphopeptides in the Fmoc mode. Int J Pept Res Ther 13:447–468CrossRefGoogle Scholar
  47. 47.
    Anthis NJ, Clore GM (2013) Sequence-specific determination of protein and peptide concentrations by absorbance at 205 nm. Protein Sci 22:851–858CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Goldfarb AR, Saidel LJ, Mosovich E (1951) The ultraviolet absorption spectra of proteins. J Biol Chem 193:397–404PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of ColoradoBoulderUSA

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