Nuclear Magnetic Resonance Spectroscopy

  • Thomas C. PochapskyEmail author
  • Susan Sondej Pochapsky
Part of the Biophysics for the Life Sciences book series (BIOPHYS, volume 6)


Nuclear magnetic resonance (NMR) has developed into an important tool for investigating the structure and dynamics of biomacromolecules in solution, associated with membranes and in solids. This chapter provides an introduction to the theory of NMR and a description of basic concepts (excitation of NMR transitions, spin populations and coherence, relaxation phenomena, signal detection and processing). Types of structural and dynamic information available from NMR experiments are noted. Standard experiments used for sequential assignment of resonances in biomolecules in solution and solid state are discussed, along with instrumentation and sample requirements. In particular, the need for selective and uniform isotope labeling is detailed. Software used to process NMR data and generate structural and dynamic information are noted, and data needed for structure determinations and dynamic analysis outlined.


Stable isotopes TROSY Nuclear Overhauser effect Dipolar coupling Solid-state NMR Protein Nucleic acid Deuteration 



Both authors acknowledge partial support from NIH grant R01-GM44191.


  1. 1.
    Pochapsky TC, Pochapsky SS (2007) NMR for physical and biological scientists. Taylor and Francis, New York, LondonGoogle Scholar
  2. 2.
    Hanson LG (2008) Is quantum mechanics necessary for understanding magnetic resonance? Concepts Magn Reson 32A:329–340CrossRefGoogle Scholar
  3. 3.
    Jeener J (1971) Lecture notes from Ampere Summer School in Basko Polje, Yugoslavia, September, 1971. In: Porneuf M, Goldman M (eds) Ampere International Summer School. Les editions de physique, Basko Polje, YugoslaviaGoogle Scholar
  4. 4.
    Kupce E, Freeman R (2008) Molecular structure from a single NMR experiment. J Am Chem Soc 130:10788–10792PubMedCrossRefGoogle Scholar
  5. 5.
    Kupce E, Freeman R, John BK (2006) Parallel acquisition of two-dimensional NMR spectra of several nuclear species. J Am Chem Soc 128:9606–9607PubMedCrossRefGoogle Scholar
  6. 6.
    Chakraborty S, Paul S, Hosur RV (2012) Simultaneous acquisition of 13C(alpha)-15N and 1H-15N-15N sequential correlations in proteins: application of dual receivers in 3D HNN. J Biomol NMR 52:5–10PubMedCrossRefGoogle Scholar
  7. 7.
    Wehrli FW, Marchand AP, Wehrli S (1988) Interpretation of carbon-13 spectra. Wiley, New YorkGoogle Scholar
  8. 8.
    Bertini I, Luchinat C (1986) NMR of paramagnetic molecules in biological systems. Benjamin-Cummings, Menlo Park, CAGoogle Scholar
  9. 9.
    Telser J (2003) Paramagnetic resonance of metallobiomolecules. American Chemical Society, Washington, DCCrossRefGoogle Scholar
  10. 10.
    Zhang W, Pochapsky SS, Pochapsky TC, Jain NU (2008) Solution NMR structure of putidaredoxin-cytochrome P450cam complex via a combined residual dipolar coupling-spin labeling approach suggests a role for Trp106 of putidaredoxin in complex formation. J Mol Biol 384:349–363PubMedCrossRefGoogle Scholar
  11. 11.
    Wang W, Perovic I, Chittuluru J, Kaganovich A, Nguyen LTT, Liao JL, Auclair JR, Johnson D, Landeru A, Simorellis AK, Ju SL, Cookson MR, Asturias FJ, Agar JN, Webb BN, Kang CH, Ringe D, Petsko GA, Pochapsky TC, Hoang QQ (2011) A soluble alpha-synuclein construct forms a dynamic tetramer. Proc Natl Acad Sci U S A 108:17797–17802PubMedCrossRefGoogle Scholar
  12. 12.
    Yuan T, Ouyang H, Vogel HJ (1999) Surface exposure of the methionine side chains of calmodulin in solution—a nitroxide spin label and two-dimensional NMR study. J Biol Chem 274:8411–8420PubMedCrossRefGoogle Scholar
  13. 13.
    Liang BY, Bushweller JH, Tamm LK (2006) Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy. J Am Chem Soc 128:4389–4397PubMedCrossRefGoogle Scholar
  14. 14.
    Zhu GA, Renwick A, Bax A (1994) Measurement of 2-Bond and 3-Bond 1H-13C J-couplings from quantitative heteronuclear J-correlation for molecules with overlapping 1H resonances, using t1 noise reduction. J Magn Reson A 110:257–261CrossRefGoogle Scholar
  15. 15.
    Schmidt JM, Sorensen OW, Ernst RR (1994) Measurement of homonuclear long-range J-Couplings by relayed E- COSY. J Magn Reson A 109:80–89CrossRefGoogle Scholar
  16. 16.
    Cordier F, Nisius L, Dingley AJ, Grzesiek S (2008) Direct detection of N-H[…]O = C hydrogen bonds in biomolecules by NMR spectroscopy. Nat Protoc 3:235–241PubMedCrossRefGoogle Scholar
  17. 17.
    Kumar A, Wagner G, Ernst RR, Wüthrich K (1980) Studies of J-connectivities and selective 1H-1H Overhauser effects in H2O solutions of biological macromolecules by two-dimensional NMR experiments. Biochem Biophys Res Commun 96:1156–1163PubMedCrossRefGoogle Scholar
  18. 18.
    Rance M, Sorensen OW, Bodenhausen G, Wagner G, Ernst RR, Wüthrich K (1983) Improved spectral resolution in COSY 1H-NMR spectra of proteins via double quantum filtering. Biochem Biophys Res Commun 117:479–485PubMedCrossRefGoogle Scholar
  19. 19.
    Braunschweiler L, Ernst RR (1983) Coherence transfer by isotropic mixing—application to proton correlation spectroscopy. J Magn Reson 53:521–528Google Scholar
  20. 20.
    Davis DG, Bax A (1985) Assignment of complex 1H-NMR spectra via two-dimensional homonuclear Hartmann–Hahn spectroscopy. J Am Chem Soc 107:2820–2821CrossRefGoogle Scholar
  21. 21.
    Bax A, Clore GM, Gronenborn AM (1990) 1H-1H correlation via isotropic mixing of 13C magnetization, a new 3-dimensional approach for assigning 1H and 13C spectra of 13C-enriched proteins. J Magn Reson 88:425–431Google Scholar
  22. 22.
    Moehle K, Freund A, Kubli E, Robinson JA (2011) NMR studies of the solution conformation of the sex peptide from Drosophila melanogaster. FEBS Lett 585:1197–1202PubMedCrossRefGoogle Scholar
  23. 23.
    Kay LE, Ikura M, Tschudin R, Bax A (1990) 3-Dimensional triple-resonance NMR spectroscopy of isotopically enriched proteins. J Magn Reson 89:496–514Google Scholar
  24. 24.
    Yamazaki T, Lee W, Revington M, Mattiello DL, Dahlquist FW, Arrowsmith CH, Kay LE (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
  25. 25.
    Salzmann M, Pervushin K, Wider G, Senn H, Wüthrich K (1999) [13C]-constant-time [15N,1H]-TROSY-HNCA for sequential assignments of large proteins. J Biomol NMR 14:85–88PubMedCrossRefGoogle Scholar
  26. 26.
    Bodenhausen G, Ruben DJ (1980) Natural abundance 15N NMR by enhanced heteronuclear spectroscopy. Chem Phys Lett 69:185CrossRefGoogle Scholar
  27. 27.
    Kay LE, Keifer P, Saarinen T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J Am Chem Soc 114:10663–10665CrossRefGoogle Scholar
  28. 28.
    Lukavsky PJ (2007) Basic principles of RNA NMR spectroscopy. In: Puglisi JD (ed) Structure and biophysics—new technologies for current challenges in biology and beyond. Springer, New York, pp 65–80CrossRefGoogle Scholar
  29. 29.
    Fiala R, Czernek J, Sklenar V (2000) Transverse relaxation optimized triple-resonance NMR experiments for nucleic acids. J Biomol NMR 16:291–302PubMedCrossRefGoogle Scholar
  30. 30.
    Patel DJ (1997) Structural analysis of nucleic acid aptamers. Curr Opin Chem Biol 1:32–46PubMedCrossRefGoogle Scholar
  31. 31.
    Pardi A (1995) Multidimensional heteronuclear NMR experiments for structure determination of isotopically labeled RNA. Methods Enzymol 261:350–380PubMedCrossRefGoogle Scholar
  32. 32.
    Kumar A, Ernst RR, Wüthrich K (1980) A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun 95:1–6PubMedCrossRefGoogle Scholar
  33. 33.
    Marion D, Kay LE, Sparks SW, Torchia DA, Bax A (1989) 3-Dimensional heteronuclear NMR of 15N-labeled proteins. J Am Chem Soc 111:1515–1517CrossRefGoogle Scholar
  34. 34.
    Ikura M, Kay LE, Tschudin R, Bax A (1990) 3-Dimensional NOESY-HMQC spectroscopy of a 13C-labeled protein. J Magn Reson 86:204–209Google Scholar
  35. 35.
    Neuhaus D, Williamson MP (2000) The nuclear Overhauser effect in structural and conformational analysis, 2nd edn. Wiley, New YorkGoogle Scholar
  36. 36.
    Noggle JH, Schirmer RE (1971) The nuclear Overhauser effect: chemical applications. Academic, New YorkGoogle Scholar
  37. 37.
    Bothner-By AA, Stephens RL, Lee JM, Warren CD, Jeanloz RW (1984) Structure determination of a tetrasaccharide—transient nuclear Overhauser effects in the rotating frame. J Am Chem Soc 106:811–813CrossRefGoogle Scholar
  38. 38.
    Bax A, Davis DG (1985) Practical aspects of two-dimensional transverse NOE spectroscopy. J Magn Reson 63:207–213Google Scholar
  39. 39.
    Tjandra N, Tate S, Ono A, Kainosho M, Bax A (2000) The NMR structure of a DNA dodecamer in an aqueous dilute liquid crystalline phase. J Am Chem Soc 122:6190–6200CrossRefGoogle Scholar
  40. 40.
    Prestegard JH, Bougault CM, Kishore AI (2004) Residual dipolar couplings in structure determination of biomolecules. Chem Rev 104:3519–3540PubMedCrossRefGoogle Scholar
  41. 41.
    Hansen MR, Mueller L, Pardi A (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Biol 5:1065–1074PubMedCrossRefGoogle Scholar
  42. 42.
    Clore GM, Starich MR, Gronenborn AM (1998) Measurement of residual dipolar couplings of macromolecules aligned in the nematic phase of a colloidal suspension of rod-shaped viruses. J Am Chem Soc 120:10571–10572CrossRefGoogle Scholar
  43. 43.
    Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114PubMedCrossRefGoogle Scholar
  44. 44.
    Wang H, Eberstadt M, Olejniczak ET, Meadows RP, Fesik SW (1998) A liquid crystalline medium for measuring residual dipolar couplings over a wide range of temperatures. J Biomol NMR 12:443–446CrossRefGoogle Scholar
  45. 45.
    Prosser RS, Losonczi JA, Shiyanovskaya IV (1998) Use of a novel aqueous liquid crystalline medium for high-resolution NMR of macromolecules in solution. J Am Chem Soc 120:11010–11011CrossRefGoogle Scholar
  46. 46.
    Ottiger M, Bax A (1999) Bicelle-based liquid crystals for NMR measurement of dipolar couplings at acidic and basic pH values. J Biomol NMR 13:187–191PubMedCrossRefGoogle Scholar
  47. 47.
    Ottiger M, Bax A (1998) Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules. J Biomol NMR 12:361–372PubMedCrossRefGoogle Scholar
  48. 48.
    Gaponenko V, Dvoretsky A, Walsby C, Hoffman BM, Rosevear PR (2000) Calculation of z-coordinates and orientational restraints using a metal binding tag. Biochemistry 39:15217–15224PubMedCrossRefGoogle Scholar
  49. 49.
    Tolman JR, Flanagan JM, Kennedy MA, Prestegard JH (1995) Nuclear magnetic dipole interactions in field-oriented proteins—information for structure determination in solution. Proc Natl Acad Sci U S A 92:9279–9283PubMedCrossRefGoogle Scholar
  50. 50.
    Tjandra N, Grzesiek S, Bax A (1996) Magnetic field dependence of nitrogen-proton J splittings in 15N-enriched human ubiquitin resulting from relaxation interference and residual dipolar coupling. J Am Chem Soc 118:6264–6272CrossRefGoogle Scholar
  51. 51.
    Purcell EM, Torrey HC, Pound RV (1946) Resonance absorption by nuclear magnetic moments in a solid. Phys Rev 69:37–38CrossRefGoogle Scholar
  52. 52.
    Gullion T, Schaefer J (1989) Rotational-echo double resonance NMR. J Magn Reson 81:196–200Google Scholar
  53. 53.
    Xu Y, Lorieau J, McDermott AE (2010) Triosephosphate isomerase: 15N and 13C chemical shift assignments and conformational change upon ligand binding by magic-angle spinning solid-state NMR spectroscopy. J Mol Biol 397:233–248PubMedCrossRefGoogle Scholar
  54. 54.
    Habenstein B, Wasmer C, Bousset L, Sourigues Y, Schuetz A, Loquet A, Meier BH, Melki R, Boeckmann A (2011) Extensive de novo solid-state NMR assignments of the 33 kDa C-terminal domain of the Ure2 prion. J Biomol NMR 51:235–243PubMedCrossRefGoogle Scholar
  55. 55.
    Takegoshi K, Nakamura S, Terao T (2001) 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637CrossRefGoogle Scholar
  56. 56.
    Gullion T (2009) Recent applications of REDOR to biological problems. In: Webb GA (ed) Annual report on NMR spectroscopy, vol 65., pp 111–137Google Scholar
  57. 57.
    Pauli J, van Rossum B, Forster H, de Groot HJM, Oschkinat H (2000) Sample optimization and identification of signal patterns of amino acid side chains in 2D RFDR spectra of the alpha-spectrin SH3 domain. J Magn Reson 143:411–416PubMedCrossRefGoogle Scholar
  58. 58.
    Demers JP, Chevelkov V, Lange A (2011) Progress in correlation spectroscopy at ultra-fast magic-angle spinning: basic building blocks and complex experiments for the study of protein structure and dynamics. Solid State Nucl Magn Reson 40:101–113PubMedCrossRefGoogle Scholar
  59. 59.
    Amoureux JP, Hu B, Trebosc J (2008) Enhanced resolution in proton solid-state NMR with very fast MAS experiments. J Magn Reson 193:305–307PubMedCrossRefGoogle Scholar
  60. 60.
    Williamson MP, Havel TF, Wüthrich K (1985) Solution conformation of proteinase inhibitor-IIa from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry. J Mol Biol 182:295–315PubMedCrossRefGoogle Scholar
  61. 61.
    Wüthrich K (1986) NMR of proteins and nucleic acids, vol 3. Wiley, New YorkGoogle Scholar
  62. 62.
    Asciutto EK, Young MJ, Madura JD, Pochapsky SS, Pochapsky TC (2012) Solution structural ensembles of substrate-free cytochrome P450(cam). Biochemistry 51:3383–3393PubMedCrossRefGoogle Scholar
  63. 63.
    Asciutto EK, Dang M, Pochapsky SS, Madura JD, Pochapsky TC (2011) Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450(cam). Biochemistry 50:1664–1671PubMedCrossRefGoogle Scholar
  64. 64.
    Grishaev A, Tugarinov V, Kay LE, Trewhella J, Bax A (2008) Refined solution structure of the 82-kDa enzyme malate synthase G from joint NMR and synchrotron SAXS restraints. J Biomol NMR 40:95–106PubMedCrossRefGoogle Scholar
  65. 65.
    Evenas J, Mittermaier A, Yang DW, Kay LE (2001) Measurement of 13C(alpha)-13C(beta) dipolar couplings in 15N,13C,2H-labeled proteins: application to domain orientation in maltose binding protein. J Am Chem Soc 123:2858–2864PubMedCrossRefGoogle Scholar
  66. 66.
    Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci U S A 94:12366–12371PubMedCrossRefGoogle Scholar
  67. 67.
    Riek R, Wider G, Pervushin K, Wüthrich K (1999) Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc Natl Acad Sci U S A 96:4918–4923PubMedCrossRefGoogle Scholar
  68. 68.
    Pielak GJ, Tian F (2012) Membrane proteins, magic-angle spinning, and in-cell NMR. Proc Natl Acad Sci U S A 109:4715–4716PubMedCrossRefGoogle Scholar
  69. 69.
    Zhou YP, Cierpicki T, Jimenez RHF, Lukasik SM, Ellena JF, Cafiso DS, Kadokura H, Beckwith J, Bushweller JH (2008) NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation. Mol Cell 31:896–908PubMedCrossRefGoogle Scholar
  70. 70.
    Gautier A, Mott HR, Bostock MJ, Kirkpatrick JP, Neitlispach D (2010) Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat Struct Mol Biol 17:768–U147PubMedCrossRefGoogle Scholar
  71. 71.
    Shi R, Proteau A, Villarroya M, Moukadiri I, Zhang L, Trempe J-F, Matte A, Armengod ME, Cygler M (2010) Structural basis for Fe-S cluster assembly and tRNA thiolation mediated by IscS protein-protein interactions. PLoS Biol 8:e1000354PubMedCrossRefGoogle Scholar
  72. 72.
    Kim JH, Tonelli M, Markley JL (2012) Disordered form of the scaffold protein IscU is the substrate for iron-sulfur cluster assembly on cysteine desulfurase. Proc Natl Acad Sci U S A 109:454–459PubMedCrossRefGoogle Scholar
  73. 73.
    Rezaei-Ghaleh N, Blackledge M, Zweckstetter M (2012) Intrinsically disordered proteins: from sequence and conformational properties toward drug discovery. Chembiochem 13:930–950PubMedCrossRefGoogle Scholar
  74. 74.
    Torchia DA (2011) Dynamics of biomolecules from picoseconds to seconds at atomic resolution. J Magn Reson 212:1–10PubMedCrossRefGoogle Scholar
  75. 75.
    Morris KF, Johnson CS (1993) Resolution of discrete and continuous molecular-size distributions by means of diffusion-ordered 2D NMR spectroscopy. J Am Chem Soc 115:4291–4299CrossRefGoogle Scholar
  76. 76.
    OuYang B, Pochapsky SS, Dang M, Pochapsky TC (2008) A functional proline switch in cytochrome P450(cam). Structure 16:916–923PubMedCrossRefGoogle Scholar
  77. 77.
    Pochapsky TC, Sligar SG, Mclachlan SJ, Lamar GN (1990) Relationship between heme binding-site structure and heme orientations of two ferrocytochrome b5s—a study in prosthetic group recognition. J Am Chem Soc 112:5258–5263CrossRefGoogle Scholar
  78. 78.
    Wei JY, Pochapsky TC, Pochapsky SS (2005) Detection of a high-barrier conformational change in the active site of cytochrome P450(cam) upon binding of putidaredoxin. J Am Chem Soc 127:6974–6976PubMedCrossRefGoogle Scholar
  79. 79.
    Bosco DA, Eisenmesser EZ, Pochapsky S, Sundquist WI, Kern D (2002) Catalysis of cis/trans isomerization in native HIV-1 capsid by human cyclophilin A. Proc Natl Acad Sci U S A 99:5247–5252PubMedCrossRefGoogle Scholar
  80. 80.
    Peng JW, Wagner G (1992) Mapping of spectral density functions using heteronuclear NMR relaxation measurements. J Magn Reson 98:308–332Google Scholar
  81. 81.
    Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J Am Chem Soc 104:4546–4559CrossRefGoogle Scholar
  82. 82.
    Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691CrossRefGoogle Scholar
  83. 83.
    Deverell C, Morgan RE, Strange JH (1970) Studies of chemical exchange by nuclear magnetic relaxation in the rotating frame. Mol Phys 18:553CrossRefGoogle Scholar
  84. 84.
    Van Horn WD, Kim HJ, Ellis CD, Hadziselimovic A, Sulistijo ES, Karra MD, Tian C, Sonnichsen FD, Sanders CR (2009) Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase. Science 324:1726–1729PubMedCrossRefGoogle Scholar
  85. 85.
    Kijac AZ, Li Y, Sligar SG, Rienstra CM (2007) Magic-angle spinning solid-state NMR Spectroscopy of nanodisc-embedded human CYP3A4. Biochemistry 46:13696–13703PubMedCrossRefGoogle Scholar
  86. 86.
    Laroche Y, Storme V, Demeutter J, Messens J, Lauwereys M (1994) High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in methylotrophic yeast Pichia pastoris. Biotechnology 12:1119–1124PubMedCrossRefGoogle Scholar
  87. 87.
    Strauss A, Bitsch F, Fendrich G, Graff P, Knecht R, Meyhack B, Jahnke W (2005) Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J Biomol NMR 31:343–349PubMedCrossRefGoogle Scholar
  88. 88.
    Lohr F, Reckel S, Karbyshev M, Connolly PJ, Abdul-Manan N, Bernhard F, Moore JM, Dotsch V (2012) Combinatorial triple-selective labeling as a tool to assist membrane protein backbone resonance assignment. J Biomol NMR 52:197–210PubMedCrossRefGoogle Scholar
  89. 89.
    Sobhanifar S, Reckel S, Junge F, Schwarz D, Kai L, Karbyshev M, Lohr F, Bernhard F, Dotsch V (2010) Cell-free expression and stable isotope labelling strategies for membrane proteins. J Biomol NMR 46:33–43PubMedCrossRefGoogle Scholar
  90. 90.
    Lustbader JW, Birken S, Pollak S, Pound A, Chait BT, Mirza UA, Ramnarain S, Canfield RE, Brown JM (1996) Expression of human chorionic gonadotropin uniformly labeled with NMR isotopes in Chinese hamster ovary cells: an advance toward rapid determination of glycoprotein structures. J Biomol NMR 7:295–304PubMedCrossRefGoogle Scholar
  91. 91.
    Pochapsky SS, Dang M, OuYang B, Simorellis AK, Pochapsky TC (2009) Redox-dependent dynamics in cytochrome P450(cam). Biochemistry 48:4254–4261PubMedCrossRefGoogle Scholar
  92. 92.
    Zwahlen C, Vincent SJF, Gardner KH, Kay LE (1998) Significantly improved resolution for NOE correlations from valine and isoleucine (C-Gamma 2) methyl groups in 15N, 13C- and 15N,13C,2H-labeled proteins. J Am Chem Soc 120:4825–4831CrossRefGoogle Scholar
  93. 93.
    Neri D, Szyperski T, Otting G, Senn H, Wüthrich K (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional C- 13 labeling. Biochemistry 28:7510–7516PubMedCrossRefGoogle Scholar
  94. 94.
    Pochapsky SS, Pochapsky TC, Wei JW (2003) A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple. Biochemistry 42:5649–5656PubMedCrossRefGoogle Scholar
  95. 95.
    Cellitti SE, Jones DH, Lagpacan L, Hao XS, Zhang Q, Hu HY, Brittain SM, Brinker A, Caldwell J, Bursulaya B, Spraggon G, Brock A, Ryu Y, Uno T, Schultz PG, Geierstanger BH (2008) In vivo incorporation of unnatural amino acids to probe structure, dynamics, and ligand binding in a large protein by nuclear magnetic resonance spectroscopy. J Am Chem Soc 130:9268–9281PubMedCrossRefGoogle Scholar
  96. 96.
    Westler WM, Stockman BJ, Markley JL (1988) Correlation of 13C and 15N chemical shifts in selectively and uniformly labeled proteins by heteronuclear two-dimensional NMR spectroscopy. J Am Chem Soc 110:6256–6258PubMedCrossRefGoogle Scholar
  97. 97.
    Chan TM, Markley JL (1983) Nuclear magnetic resonance studies of 2-iron-2-sulfur ferredoxins.3. Heteronuclear (13C,1H) two-dimensional NMR spectra, 13C peak assignments, and 13C relaxation measurements. Biochemistry 22:5996–6002CrossRefGoogle Scholar
  98. 98.
    Jain NU, Pochapsky TC (1998) Redox dependence of hyperfine-shifted 13C and 15N resonances in putidaredoxin. J Am Chem Soc 120:12984–12985CrossRefGoogle Scholar
  99. 99.
    Kobayashi H, Swapna GVT, Wu KP, Afinogenova Y, Conover K, Mao BC, Montelione GT, Inouye M (2012) Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility. J Biomol NMR 52:303–313PubMedCrossRefGoogle Scholar
  100. 100.
    Bockmann A, Lange A, Galinier A, Luca S, Giraud N, Juy M, Heise H, Montserret R, Penin F, Baldus M (2003) Solid state NMR sequential resonance assignments and conformational analysis of the 2 × 10.4 kDa dimeric form of the Bacillus subtilis protein Crh. J Biomol NMR 27:323–339PubMedCrossRefGoogle Scholar
  101. 101.
    Kim Y, Valentine K, Opella SJ, Schendel SL, Cramer WA (1998) Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers. Protein Sci 7:342–348PubMedCrossRefGoogle Scholar
  102. 102.
    Mote KR, Gopinath T, Traaseth NJ, Kitchen J, Gor’kov PL, Brey WW, Veglia G (2011) Multidimensional oriented solid-state NMR experiments enable the sequential assignment of uniformly 15N labeled integral membrane proteins in magnetically aligned lipid bilayers. J Biomol NMR 51:339–346PubMedCrossRefGoogle Scholar
  103. 103.
    Kazanis S, Pochapsky TC (1997) Structural features of the metal binding site and dynamics of gallium putidaredoxin, a diamagnetic derivative of a Cys4Fe2S2 ferredoxin. J Biomol NMR 9:337–346PubMedCrossRefGoogle Scholar
  104. 104.
    Piotto M, Saudek V, Sklenar V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2:661–665PubMedCrossRefGoogle Scholar
  105. 105.
    Kuboniwa H, Grzesiek S, Delaglio F, Bax A (1994) Measurement of H-N-H-alpha J couplings in calcium-free calmodulin using new 2D and 3D water flip-back methods. J Biomol NMR 4:871–878PubMedCrossRefGoogle Scholar
  106. 106.
    Grzesiek S, Bax A (1993) The importance of not daturating H2O in protein NMR—application to sensitivity enhancement and NOE measurements. J Am Chem Soc 115:12593–12594CrossRefGoogle Scholar
  107. 107.
    Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C, and 15N spectra of larger proteins - Heteronuclear triple-resonance 3-dimensional NMR spectroscopy—application to calmodulin. Biochemistry 29:4659–4667PubMedCrossRefGoogle Scholar
  108. 108.
    Bermel W, Bertini I, Felli IC, Matzapetakis M, Pierattelli R, Theli EC, Turano P (2007) A method for C-alpha direct detection in protonless NMR. J Magn Reson 188:301–310PubMedCrossRefGoogle Scholar
  109. 109.
    Bermel W, Bertini I, Duma L, Felli IC, Emsley L, Pierattelli R, Vasos PR (2005) Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew Chem Int Ed 44:3089–3092CrossRefGoogle Scholar
  110. 110.
    Kostic M, Pochapsky SS, Pochapsky TC (2002) Rapid recycle 13C′,15N and 13C,13C′ heteronuclear and homonuclear multiple quantum coherence detection for resonance assignments in paramagnetic proteins: example of Ni2+-containing acireductone dioxygenase. J Am Chem Soc 124:9054–9055PubMedCrossRefGoogle Scholar
  111. 111.
    Pochapsky SS, Sunshine JC, Pochapsky TC (2008) Completing the circuit: direct-observe 13C,15N double-quantum spectroscopy permits sequential resonance assignments near a paramagnetic center in acireductone dioxygenase. J Am Chem Soc 130:2156PubMedCrossRefGoogle Scholar
  112. 112.
    Machonkin TE, Westler WM, Markley JL (2002) 13C{13C–2D NMR: a novel strategy for the study of paramagnetic proteins with slow electronic relaxation rates. J Am Chem Soc 124:3204–3205PubMedCrossRefGoogle Scholar
  113. 113.
    Matzapetakis M, Turano P, Theil EC, Bertini I (2007) 13C-13C NOESY spectra of a 480 kDa protein: solution NMR of ferritin. J Biomol NMR 38:237–242PubMedCrossRefGoogle Scholar
  114. 114.
    Szyperski T, Fernandez C, Ono A, Wüthrich K, Kainosho M (1999) The 2D 31P spin-echo-difference constant-time [13C, 1H]-HMQC experiment for simultaneous determination of 3J(H3′P) and 3J(C4′P) in 13C-labeled nucleic acids and their protein complexes. J Magn Reson 140:491–494PubMedCrossRefGoogle Scholar
  115. 115.
    Riek R, Pervushin K, Fernandez C, Kainosho M, Wüthrich K (2001) [13C,13C]- and [13C,1H]-TROSY in a triple resonance experiment for ribose base and intrabase correlations in nucleic acids. J Am Chem Soc 123:658–664PubMedCrossRefGoogle Scholar
  116. 116.
    Ding K, Gronenborn AM (2004) Sensitivity-enhanced IPAP experiments for measuring one-bond 13C′-13C(alpha) and 13C(alpha)-1H(alpha) residual dipolar couplings in proteins. J Magn Reson 167:253–258PubMedCrossRefGoogle Scholar
  117. 117.
    Ding KY, Gronenborn AM (2003) Sensitivity-enhanced 2D IPAP, TROSY-anti-TROSY, and E.COSY experiments: alternatives for measuring dipolar 15N-1H(N) couplings. J Magn Reson 163:208–214PubMedCrossRefGoogle Scholar
  118. 118.
    Hahn EL (1950) Spin echos. Phys Rev 80:580–594CrossRefGoogle Scholar
  119. 119.
    Stejskal EO, Tanner JE (1965) Spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient. J Chem Phys 42:288–292CrossRefGoogle Scholar
  120. 120.
    Scaglioni L, Mazzini S, Mondelli R, Dallavalle S, Gattinoni S, Tinelli S, Beretta GL, Zunino F, Ragg E (2009) Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison. Bioorg Med Chem 17:484–491PubMedCrossRefGoogle Scholar
  121. 121.
    Mizukoshi Y, Abe A, Takizawa T, Hanzawa H, Fukunishi Y, Shimada I, Takahashi H (2012) An accurate pharmacophore mapping method by NMR spectroscopy. Angew Chem Int Ed 51:1362–1365CrossRefGoogle Scholar
  122. 122.
    Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRpipe—a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293PubMedCrossRefGoogle Scholar
  123. 123.
    NMR Felix (2007) Felix. Felix NMR Inc., San Diego, CAGoogle Scholar
  124. 124.
    Hare DR (1986) FTNMR. Hare Research, Woodinville, WAGoogle Scholar
  125. 125.
    Bartels C, Xia TH, Billeter M, Guntert P, Wüthrich K (1995) The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J Biomol NMR 6:1–10PubMedCrossRefGoogle Scholar
  126. 126.
    Bettendorff P, Damberger F, Rochus L, Keller J, Guntert P CARA (computer assisted resonance assignment). ETH, ZurichGoogle Scholar
  127. 127.
    Goddard TD, Kneller DG Sparky 3. UCSF, San FranciscoGoogle Scholar
  128. 128.
    NMRView. One Moon Scientific, Inc., Newark, NJGoogle Scholar
  129. 129.
    Moseley HNB, Sperling LJ, Rienstra CM (2010) Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of beta 1 immunoglobulin binding domain of protein G (GB1). J Biomol NMR 48:123–128PubMedCrossRefGoogle Scholar
  130. 130.
    O’connell JF, Pryer KD, Grant SK, Leiting B (1999) A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: application of an automated assignment strategy using GARANT. J Biomol NMR 13:311–324PubMedCrossRefGoogle Scholar
  131. 131.
    Crippen GM, Rousaki A, Revington M, Zhang YB, Zuiderweg ERP (2010) SAGA: rapid automatic mainchain NMR assignment for large proteins. J Biomol NMR 46:281–298PubMedCrossRefGoogle Scholar
  132. 132.
    Zweckstetter M (2003) Determination of molecular alignment tensors without backbone resonance assignment: aid to rapid analysis of protein-protein interactions. J Biomol NMR 27:41–56PubMedCrossRefGoogle Scholar
  133. 133.
    Guerry P, Herrmann T (2011) Advances in automated NMR protein structure determination. Q Rev Biophys 44:257–309PubMedCrossRefGoogle Scholar
  134. 134.
    Wagner G, Braun W, Havel TF, Schaumann T, Go N, Wüthrich K (1987) Protein structures in solution by nuclear magnetic resonance and distance geometry—the polypeptide fold of the basic pancreatic trypsin inhibitor determined using 2 different algorithms, DISGEO and DISMAN. J Mol Biol 196:611–639PubMedCrossRefGoogle Scholar
  135. 135.
    Case DA, Cheatham TE, Darden T, Gohlke H, Luo R, Merz KM, Onufriev A, Simmerling C, Wang B, Woods RJ (2005) The AMBER biomolecular simulation programs. J Comput Chem 26:1668–1688PubMedCrossRefGoogle Scholar
  136. 136.
    Schwieters CD, Kuszewski JJ, Tjandra N, Clore GM (2003) The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160:65–73PubMedCrossRefGoogle Scholar
  137. 137.
    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14:33–38PubMedCrossRefGoogle Scholar
  138. 138.
    Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54:905–921PubMedCrossRefGoogle Scholar
  139. 139.
    Guntert P (2004) Automated NMR protein structure calculation with CYANA. Methods Mol Biol 278:353–378PubMedGoogle Scholar
  140. 140.
    Banci L, Bertini I, Cremonini MA, Gori-Savellini G, Luchinat C, Wüthrich K, Guntert P (1998) Pseudyana for NMR structure calculation of paramagnetic metalloproteins using torsion angle molecular dynamics. J Biomol NMR 12:553–557PubMedCrossRefGoogle Scholar
  141. 141.
    Guntert P, Mumenthaler C, Wüthrich K (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol 273:283–298PubMedCrossRefGoogle Scholar
  142. 142.
    Gorlach M, Wittekind M, Farmer BT, Kay LE, Mueller L (1993) Measurement of 3J(HN-Alpha) vicinal coupling constants in proteins. J Magn Reson B 101:194–197CrossRefGoogle Scholar
  143. 143.
    Mollova ET, Pardi A (2000) NMR solution structure determination of RNAs. Curr Opin Struct Biol 10:298–302PubMedCrossRefGoogle Scholar
  144. 144.
    Shen Y, Delaglio F, Bax A (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223PubMedCrossRefGoogle Scholar
  145. 145.
    Case DA (1998) The use of chemical shifts and their anisotropies in biomolecular structure determination. Curr Opin Struct Biol 8:624–630PubMedCrossRefGoogle Scholar
  146. 146.
    Valafar H, Prestegard JH (2004) REDCAT: a residual dipolar coupling analysis tool. J Magn Reson 167:228–241PubMedCrossRefGoogle Scholar
  147. 147.
    Zweckstetter M (2008) NMR: prediction of molecular alignment from structure using the PALES software. Nat Protoc 3:679–690PubMedCrossRefGoogle Scholar
  148. 148.
    Fedyukina DV, Cavagnero S (2011) Protein folding at the exit tunnel. In: Rees DC, Dill KA, Williamson JR (eds) Annual review on biophysics, vol 40., pp 337–359Google Scholar
  149. 149.
    Cioni P, Gabellieri E (2011) Protein dynamics and pressure: what can high pressure tell us about protein structural flexibility? Biochim Biophys Acta 1814:934–941PubMedCrossRefGoogle Scholar
  150. 150.
    Cozzolino S, Sequi P, Valentini M (2011) Probing interactions between small molecules and polymers by means of NMR spectroscopy. In: Webb GA (ed) Annual report on NMR spectroscopy, vol 74., pp 181–213Google Scholar
  151. 151.
    Manley G, Loria JP (2012) NMR insights into protein allostery. Arch Biochem Biophys 519:223–231PubMedCrossRefGoogle Scholar
  152. 152.
    Bhunia A, Bhattacharjya S, Chatterjee S (2012) Applications of saturation transfer difference NMR in biological systems. Drug Discov Today 17:505–513PubMedCrossRefGoogle Scholar
  153. 153.
    Barelier S, Krimm I (2011) Ligand specificity, privileged substructures and protein druggability from fragment-based screening. Curr Opin Chem Biol 15:469–474PubMedCrossRefGoogle Scholar
  154. 154.
    Sattler M, Schleucher J, Griesinger C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients. Prog Nucl Magn Reson Spectrosc 34:93–158CrossRefGoogle Scholar
  155. 155.
    Ippel JH, Wijmenga SS, de Jong R, Heus HA, Hilbers CW, de Vroom E, van der Marel GA, van Boom JH (1996) Heteronuclear scalar couplings in the bases and sugar rings of nucleic acids: their determination and application in assignment and conformational analysis. Magn Reson Chem 34:S176CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Chemistry and Rosenstiel Basic Medical Sciences Research InstituteBrandeis UniversityWalthamUSA

Personalised recommendations