Anisotropic and Isotropic Chemical Shifts Perturbations from Solid State NMR Spectroscopy for Structural and Functional Biology

  • Eduard A. ChekmenevEmail author
  • Joana Paulino
  • Riqiang Fu
  • Timothy A. Cross
Reference work entry


A molecular structure determined by crystallography, cryo-EM, or NMR is an excellent starting point for our understanding of how biology is accomplished, but NMR is one of the best tools for going beyond this starting point for a detailed characterization of functional mechanisms, even for an understanding of kinetic rates. To this end one of the best approaches for doing this is through the observation of isotropic and anisotropic chemical shift perturbations. Two molecular systems that have been extensively studied and characterized exemplify the usefulness of chemical shift perturbation as an effective strategy for understanding functional activities. Gramicidin A, an antibiotic from Bacillus brevis, that as a dimer forms a monovalent cation channel and a protein from Influenza A virus, the M2 protein that as a tetramer forms a proton channel. Blocking the M2 proton channel is an effective anti-influenza strategy. For gramicidin it was discovered that different monovalent cations have different binding sites at the mouth and exit of the channel accounting for the different solvation energy requirements of the various cations. For M2 the functional activity of a unique histidine tetrad that shuttles protons into the viral interior through a balance of futile and conductance protonation cycles was elucidated by chemical shift perturbations.


Gramicidin A M2 proton channel Solid-state NMR 15N NMR 17O NMR Anisotropic chemical shift interactions Magic angle spinning ssNMR Cation binding site Oriented sample ssNMR Isotropic chemical shift perturbations Proton conductance 


  1. 1.
    Anfinsen CB. Principles that govern the folding of protein chains. Science. 1973;181(96):223–30.CrossRefGoogle Scholar
  2. 2.
    Anderson LC, DeHart CJ, Kaiser NK, Fellers RT, Smith DF, Greer JB, et al. Identification and characterization of human proteoforms by top-down LC-21 tesla FT-ICR mass spectrometry. J Proteome Res. 2016;16:1087–1096.CrossRefGoogle Scholar
  3. 3.
    Zhou HX, Cross TA. Influences of membrane mimetic environments on membrane protein structures. Annu Rev Biophys. 2013;42:361–92.CrossRefGoogle Scholar
  4. 4.
    Cross TA, Ekanayake V, Paulino J, Wright A. Solid state NMR: The essential technology for helical membrane protein structural characterization. J Magn Reson. 2014;239:100–9.CrossRefGoogle Scholar
  5. 5.
    Ketchem RR, Hu W, Cross TA. High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. Science. 1993;261(5127):1457–60.CrossRefGoogle Scholar
  6. 6.
    Ketchem RR, Roux B, Cross TA. High-resolution polypeptide structure in a lamellar phase lipid environment from solid-state NMR derived orientational constraints. Structure. 1997;5:1655–69.CrossRefGoogle Scholar
  7. 7.
    Hu W, Lazo ND, Cross TA. Tryptophan dynamics and structural refinement in a lipid bilayer environment: solid state NMR of the gramicidin channel. Biochemistry. 1995;34(43):14138–46.CrossRefGoogle Scholar
  8. 8.
    Cross TA, Arseniev A, Cornell BA, Davis JH, Killian JA, Koeppe RE, et al. Gramicidin channel controversy – revisited. Nat Struct Biol. 1999;6(7):610–2.CrossRefGoogle Scholar
  9. 9.
    Andersen OS, Apell H-J, Bamberg E, Busath DD, Koeppe REI, Sigworth FJ, et al. Gramicidin channel controversy – the structure in a lipid environment. Nat Struct Biol. 1999;6:609.CrossRefGoogle Scholar
  10. 10.
    Arseniev AS, Barsukov IL, Bystrov VF, Lomize AL, Ovchinnikov YA. 1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices. FEBS Lett. 1985;186(2):168–74.CrossRefGoogle Scholar
  11. 11.
    Burkhart BM, Li N, Langs DA, Pangborn WA, Duax WL. The conducting form of gramicidin A is a right-handed double-stranded double helix. Proc Natl Acad Sci U S A. 1998;95(22):12950–5.CrossRefGoogle Scholar
  12. 12.
    Hofer N, Aragao D, Caffrey M. Crystallizing transmembrane peptides in lipidic mesophases. Biophys J. 2010;99(3):L23–5.CrossRefGoogle Scholar
  13. 13.
    Miao Y, Fu R, Zhou HX, Cross TA. Dynamic short hydrogen bonds in histidine tetrad of full-length M2 proton channel reveal tetrameric structural heterogeneity and functional mechanism. Structure. 2015;23(12):2300–8.CrossRefGoogle Scholar
  14. 14.
    Wang J, Kim S, Kovacs F, Cross TA. Structure of the transmembrane region of the M2 protein H(+) channel. Protein Sci. 2001;10(11):2241–50.CrossRefGoogle Scholar
  15. 15.
    Nishimura K, Kim S, Zhang L, Cross TA. The closed state of a H+ channel helical bundle: combining precise orientational and distance restraints from solid state NMR. Biochemistry. 2002;41:13170–7.CrossRefGoogle Scholar
  16. 16.
    Stouffer AL, Acharya R, Salom D, Levine AS, Di Costanzo L, Soto CS, et al. Structural basis for the function and inhibition of an influenza virus proton channel. Nature. 2008;451:596–9.CrossRefGoogle Scholar
  17. 17.
    Acharya R, Carnevale V, Fiorin G, Levine BG, Polishchuk AL, Balannik V, et al. Structure and mechanism of proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A virus. Proc Natl Acad Sci U S A. 2010;107(34):15075–80.CrossRefGoogle Scholar
  18. 18.
    Sharma M, Yi M, Dong H, Qin H, Peterson E, Busath DD, et al. Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer. Science. 2010;330(6003):509–12.CrossRefGoogle Scholar
  19. 19.
    Schnell JR, Chou JJ. Structure and mechanism of the M2 proton channel of influenza A virus. Nature. 2008;451:591–5.CrossRefGoogle Scholar
  20. 20.
    Ketchem RR, Lee KC, Huo S, Cross TA. Macromolecular structural elucidation with solid-state NMR-derived orientational constraints. J Biomol NMR. 1996;8(1):1–14.CrossRefGoogle Scholar
  21. 21.
    Lazo ND, Hu W, Lee KC, Cross TA. Rapidly-frozen polypeptide samples for characterization of high definition dynamics by solid-state NMR spectroscopy. Biochem Biophys Res Commun. 1993;197(2):904–9.CrossRefGoogle Scholar
  22. 22.
    North CL, Cross TA. Correlations between function and dynamics: time scale coincidence for ion translocation and molecular dynamics in the gramicidin channel backbone. Biochemistry. 1995;34(17):5883–95.CrossRefGoogle Scholar
  23. 23.
    Lee KC, Huo S, Cross TA. Lipid-peptide interface: valine conformation and dynamics in the gramicidin channel. Biochemistry. 1995;34(3):857–67.CrossRefGoogle Scholar
  24. 24.
    Kim S, Quine JR, Cross TA. Complete cross-validation and R-factor calculation of a solid-state NMR derived structure. J Am Chem Soc. 2001;123(30):7292–8.CrossRefGoogle Scholar
  25. 25.
    Fu R, Cotten M, Cross TA. Inter- and intramolecular distance measurements by solid-state MAS NMR: determination of gramicidin A channel dimer structure in hydrated phospholipid bilayers. J Biomol NMR. 2000;16(3):261–8.CrossRefGoogle Scholar
  26. 26.
    Tian F, Cross TA. Cation transport: an example of structural based selectivity. J Mol Biol. 1999;285(5):1993–2003.CrossRefGoogle Scholar
  27. 27.
    Tian F, Cross TA. Cation binding induced changes in 15N CSA in a membrane-bound polypeptide. J Magn Reson. 1998;135(2):535–40.CrossRefGoogle Scholar
  28. 28.
    Hinton JF, Whaley WL, Shungu D, Koeppe 2nd RE, Millett FS. Equilibrium binding constants for the group I metal cations with gramicidin-A determined by competition studies and T1+-205 nuclear magnetic resonance spectroscopy. Biophys J. 1986;50(3):539–44.CrossRefGoogle Scholar
  29. 29.
    Dzidic JaK P. Hydration of the alkali ions in the gas phase. Enthalpies and entropies of reactions M+(H2O)n-1 + H2O = M+(H2O)n. J Phys Chem. 1970;74:1466–74.CrossRefGoogle Scholar
  30. 30.
    Hu J, Chekmenev EY, Gan Z, Gor’kov PL, Saha S, Brey WW, et al. Ion solvation by channel carbonyls characterized by 17O solid-state NMR at 21T. J Am Chem Soc. 2005;127(34):11922–3.CrossRefGoogle Scholar
  31. 31.
    Fu R, Brey WW, Shetty K, Gor’kov P, Saha S, Long JR, et al. Ultra-wide bore 900 MHz high-resolution NMR at the National High Magnetic Field Laboratory. J Magn Reson. 2005;177(1):1–8.CrossRefGoogle Scholar
  32. 32.
    Chekmenev EY, Waddell KW, Hu J, Gan Z, Wittebort RJ, Cross TA. Ion-binding study by 17O solid-state NMR spectroscopy in the model peptide Gly-Gly-Gly at 19.6T. J Am Chem Soc. 2006;128(30):9849–55.CrossRefGoogle Scholar
  33. 33.
    Chekmenev EY, Gor’kov PL, Cross TA, Alaouie AM, Smirnov AI. Flow-through lipid nanotube arrays for structure-function studies of membrane proteins by solid-state NMR spectroscopy. Biophys J. 2006;91(8):3076–84.CrossRefGoogle Scholar
  34. 34.
    Jones TL, Fu R, Nielson F, Cross TA, Busath DD. Gramicidin channels are internally gated. Biophys J. 2010;98(8):1486–93.CrossRefGoogle Scholar
  35. 35.
    Li C, Qin H, Gao FP, Cross TA. Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel. Biochim Biophys Acta. 2007;1768(12):3162–70.CrossRefGoogle Scholar
  36. 36.
    Miao Y, Qin H, Fu R, Sharma M, Can TV, Hung I, et al. M2 proton channel structural validation from full-length protein samples in synthetic bilayers and E. coli membranes. Angew Chem Int Ed Eng. 2012;51:8383–6.CrossRefGoogle Scholar
  37. 37.
    Miao Y, Cross TA, Fu R. Identifying inter-residue resonances in crowded 2D C- C chemical shift correlation spectra of membrane proteins by solid-state MAS NMR difference spectroscopy. J Biomol NMR. 2013;56:265–73.CrossRefGoogle Scholar
  38. 38.
    Ekanayake EV, Fu R, Cross TA. Structural influences: cholesterol, drug, and proton binding to full-length influenza A M2 protein. Biophys J. 2016;110(6):1391–9.CrossRefGoogle Scholar
  39. 39.
    Wright AK, Batsomboon P, Dai J, Hung I, Zhou HX, Dudley GB, et al. Differential binding of rimantadine enantiomers to influenza A M2 proton channel. J Am Chem Soc. 2016;138(5):1506–9.CrossRefGoogle Scholar
  40. 40.
    Hu J, Fu R, Nishimura K, Zhang L, Zhou HX, Busath DD, et al. Histidines, heart of the hydrogen ion channel from influenza A virus: toward an understanding of conductance and proton selectivity. Proc Natl Acad Sci U S A. 2006;103(18):6865–70.CrossRefGoogle Scholar
  41. 41.
    Hong M, Fritzsching KJ, Williams JK. Hydrogen-bonding partner of the proton-conducting histidine in the influenza M2 proton channel revealed from 1H chemical shifts. J Am Chem Soc. 2012;134(36):14753–5.CrossRefGoogle Scholar
  42. 42.
    Fu R, Miao Y, Qin H, Cross TA. Probing hydronium ion histidine NH exchange rate constants in the M2 channel via indirect observation of dipolar-dephased 15N signals in magic-angle-spinning NMR. J Am Chem Soc. 2016;138(49):15801–4.CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Eduard A. Chekmenev
    • 1
    Email author
  • Joana Paulino
    • 2
    • 3
  • Riqiang Fu
    • 3
  • Timothy A. Cross
    • 2
    • 3
    • 4
  1. 1.Institute of Imaging ScienceVanderbilt UniversityNashvilleUSA
  2. 2.Institute of Molecular BiophysicsFlorida State UniversityTallahasseeUSA
  3. 3.National High Magnetic Field LaboratoryTallahasseeUSA
  4. 4.Department of Chemistry and BiochemistryFlorida State UniversityTallahasseeUSA

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