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Journal of Biomolecular NMR

, Volume 55, Issue 2, pp 189–200 | Cite as

Secondary structural analysis of proteins based on 13C chemical shift assignments in unresolved solid-state NMR spectra enhanced by fragmented structure database

Article

Abstract

Magic-angle-spinning solid-state 13C NMR spectroscopy is useful for structural analysis of non-crystalline proteins. However, the signal assignments and structural analysis are often hampered by the signal overlaps primarily due to minor structural heterogeneities, especially for uniformly-13C,15N labeled samples. To overcome this problem, we present a method for assigning 13C chemical shifts and secondary structures from unresolved two-dimensional 13C–13C MAS NMR spectra by spectral fitting, named reconstruction of spectra using protein local structures (RESPLS). The spectral fitting was conducted using databases of protein fragmented structures related to 13Cα, 13Cβ, and 13C′ chemical shifts and cross-peak intensities. The experimental 13C–13C inter- and intra-residue correlation spectra of uniformly isotope-labeled ubiquitin in the lyophilized state had a few broad peaks. The fitting analysis for these spectra provided sequence-specific Cα, Cβ, and C′ chemical shifts with an accuracy of about 1.5 ppm, which enabled the assignment of the secondary structures with an accuracy of 79 %. The structural heterogeneity of the lyophilized ubiquitin is revealed from the results. Test of RESPLS analysis for simulated spectra of five different types of proteins indicated that the method allowed the secondary structure determination with accuracy of about 80 % for the 50–200 residue proteins. These results demonstrate that the RESPLS approach expands the applicability of the NMR to non-crystalline proteins exhibiting unresolved 13C NMR spectra, such as lyophilized proteins, amyloids, membrane proteins and proteins in living cells.

Keywords

Signal assignment Secondary structures Fragment assembly Spectral simulation Signal overlap 

Notes

Acknowledgments

This work was supported by funding from the Targeted Proteins Research Program and KAKENHI of the Ministry of Education, Culture, Sports, Sciences and Technology of Japan.

Supplementary material

10858_2012_9701_MOESM1_ESM.doc (33.4 mb)
Supplementary material 1 (DOC 34,243 kb)

References

  1. Baldus M (2007) Magnetic resonance in the solid state: applications to protein folding, amyloid fibrils and membrane proteins. Eur Biophys J 36(Suppl 1):S37–S48CrossRefGoogle Scholar
  2. Baldus M, Petkova AT, Herzfeld J, Griffin RG (1998) Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol Phys 95:1197–1207ADSCrossRefGoogle Scholar
  3. Bayro MJ, Debelouchina GT, Eddy MT, Birkett NR, MacPhee CE, Rosay M, Maas WE, Dobson CM, Griffin RG (2011) Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR. J Am Chem Soc 133:13967–13974CrossRefGoogle Scholar
  4. Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG (1995) Heteronuclear decoupling in rotating solids. J Chem Phys 103:6951–6958ADSCrossRefGoogle Scholar
  5. 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 27:323–339CrossRefGoogle Scholar
  6. Bradley P, Misura KM, Baker D (2005) Toward high-resolution de novo structure prediction for small proteins. Science 309:1868–1871ADSCrossRefGoogle Scholar
  7. Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420:98–102ADSCrossRefGoogle Scholar
  8. Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13:289–302CrossRefGoogle Scholar
  9. Egawa A, Fujiwara T, Mizoguchi T, Kakitani Y, Koyama Y, Akutsu H (2007) Structure of the light-harvesting bacteriochlorophyll c assembly in chlorosomes from Chlorobium limicola determined by solid-state NMR. Proc Natl Acad Sci USA 104:790–795ADSCrossRefGoogle Scholar
  10. Franks WT, Wylie BJ, Stellfox SA, Rienstra CM (2006) Backbone conformational constraints in a microcrystalline U-15N-labeled protein by 3D dipolar-shift solid-state NMR spectroscopy. J Am Chem Soc 128:3154–3155CrossRefGoogle Scholar
  11. Fujiwara T, Todokoro Y, Yanagishita H, Tawarayama M, Kohno T, Wakamatsu K, Akutsu H (2004) Signal assignments and chemical-shift structural analysis of uniformly 13C, 15N-labeled peptide, mastoparan-X, by multidimensional solid-state NMR under magic-angle spinning. J Biomol NMR 28:311–325CrossRefGoogle Scholar
  12. Goldbourt A, Gross BJ, Day LA, McDermott AE (2007) Filamentous phage studied by magic-angle spinning NMR: resonance assignment and secondary structure of the coat protein in Pf1. J Am Chem Soc 129:2338–2344CrossRefGoogle Scholar
  13. Greenwald J, Buhtz C, Ritter C, Kwiatkowski W, Choe S, Maddelein ML, Ness F, Cescau S, Soragni A, Leitz D, Saupe SJ, Riek R (2010) The mechanism of prion inhibition by HET-S. Mol Cell 38:889–899CrossRefGoogle Scholar
  14. Habeck M, Rieping W, Nilges M (2006) Weighting of experimental evidence in macromolecular structure determination. Proc Natl Acad Sci USA 103:1756–1761ADSCrossRefGoogle Scholar
  15. Havlin RH, Tycko R (2005) Probing site-specific conformational distributions in protein folding with solid-state NMR. Proc Natl Acad Sci USA 102:3284–3289ADSCrossRefGoogle Scholar
  16. Henikoff S, Henikoff JG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 89:10915–10919ADSCrossRefGoogle Scholar
  17. Hohwy M, Jakobsen HJ, Eden M, Levitt MH, Nielsen NC (1998) Broadband dipolar recoupling in the nuclear magnetic resonance of rotating solids: a compensated C7 pulse sequence. J Chem Phys 108:2686–2694ADSCrossRefGoogle Scholar
  18. Hong M (1999) Solid-state dipolar INADEQUATE NMR spectroscopy with a large double-quantum spectral width. J Magn Reson 136:86–91ADSCrossRefGoogle Scholar
  19. Hughes CE, Luca S, Baldus M (2004) Radio-frequency driven polarization transfer without heteronuclear decoupling in rotating solids. Chem Phys Lett 385:435–440ADSCrossRefGoogle Scholar
  20. Iben IET, Braunstein D, Doster W, Frauenfelder H, Hong MK, Johnson JB, Luck S, Ormos P, Schulte A, Steinbach PJ, Xie AH, Young RD (1989) Glassy behavior of a protein. Phys Rev Lett 62:1916–1919ADSCrossRefGoogle Scholar
  21. Igumenova TI, McDermott AE (2003) Improvement of resolution in solid state NMR spectra with J-decoupling: an analysis of lineshape contributions in uniformly C-13-enriched amino acids and proteins. J Magn Reson 164:270–285ADSCrossRefGoogle Scholar
  22. Igumenova TI, McDermott AE, Zilm KW, Martin RW, Paulson EK, Wand AJ (2004) Assignments of carbon NMR resonances for microcrystalline ubiquitin. J Am Chem Soc 126:6720–6727CrossRefGoogle Scholar
  23. Ikeda K, Kameda T, Harada E, Akutsu H, Fujiwara T (2011) Combined use of replica-exchange molecular dynamics and magic-angle-spinning solid-state NMR spectral simulations for determining the structure and orientation of membrane-bound peptide. J Phys Chem B 115:9327–9336CrossRefGoogle Scholar
  24. Iwata K, Fujiwara T, Matsuki Y, Akutsu H, Takahashi S, Naiki H, Goto Y (2006) 3D structure of amyloid protofilaments of beta2-microglobulin fragment probed by solid-state NMR. Proc Natl Acad Sci USA 103:18119–18124ADSCrossRefGoogle Scholar
  25. Jakeman DL, Mitchell DJ, Shuttleworth WA, Evans JNS (1998) Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes. J Biomol NMR 12:417–421CrossRefGoogle Scholar
  26. Juy M, Penin F, Favier A, Galinier A, Montserret R, Haser R, Deutscher J, Bockmann A (2003) Dimerization of Crh by reversible 3D domain swapping induces structural adjustments to its monomeric homologue Hpr. J Mol Biol 332:767–776CrossRefGoogle Scholar
  27. Kainosho M, Torizawa T, Iwashita Y, Terauchi T, Ono AM, Guntert P (2006) Optimal isotope labelling for NMR protein structure determinations. Nature 440:52–57ADSCrossRefGoogle Scholar
  28. Kay LE (2005) NMR studies of protein structure and dynamics. J Magn Reson 173:193–207ADSCrossRefGoogle Scholar
  29. Kennedy SD, Bryant RG (1990) Structural effects of hydration—studies of lysozyme by C-13 solids NMR. Biopolymers 29:1801–1806CrossRefGoogle Scholar
  30. Khitrin AK, Fujiwara T, Akutsu H (2003) Phase-modulated heteronuclear decoupling in NMR of solids. J Magn Reson 162:46–53ADSCrossRefGoogle Scholar
  31. Kirkpatrick S, Gelatt CD Jr, Vecchi MP (1983) Optimization by simulated annealing. Science 220:671–680MathSciNetADSMATHCrossRefGoogle Scholar
  32. Kitao A, Hayward S, Go N (1998) Energy landscape of a native protein: jumping-among-minima model. Proteins 33:496–517CrossRefGoogle Scholar
  33. Kobayashi M, Matsuki Y, Yumen I, Fujiwara T, Akutsu H (2006) Signal assignment and secondary structure analysis of a uniformly [13C, 15N]-labeled membrane protein, H+-ATP synthase subunit c, by magic-angle spinning solid-state NMR. J Biomol NMR 36:279–293CrossRefGoogle Scholar
  34. Kubo A, Mcdowell CA (1988) Spectral spin diffusion in polycrystalline solids under magic-angle spinning. J Chem Soc Faraday Trans 1(84):3713–3730Google Scholar
  35. Lange A, Gattin Z, Van Melckebeke H, Wasmer C, Soragni A, van Gunsteren WF, Meier BH (2009) A combined solid-state NMR and MD characterization of the stability and dynamics of the HET-s(218–289) prion in its amyloid conformation. ChemBioChem 10:1657–1665CrossRefGoogle Scholar
  36. Long HW, Tycko R (1998) Biopolymer conformational distributions from solid-state NMR: alpha-helix and 3(10)-helix contents of a helical peptide. J Am Chem Soc 120:7039–7048CrossRefGoogle Scholar
  37. Luca S, Filippov DV, van Boom JH, Oschkinat H, de Groot HJM, Baldus M (2001) Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under Magic Angle Spinning. J Biomol NMR 20:325–331CrossRefGoogle Scholar
  38. Markley JL, Ulrich EL, Berman HM, Henrick K, Nakamura H, Akutsu H (2008) BioMagResBank (BMRB) as a partner in the Worldwide Protein Data Bank (wwPDB): new policies affecting biomolecular NMR depositions. J Biomol NMR 40:153–155CrossRefGoogle Scholar
  39. Martin RW, Zilm KW (2003) Preparation of protein nanocrystals and their characterization by solid state NMR. J Magn Reson 165:162–174ADSCrossRefGoogle Scholar
  40. Matsuki Y, Akutsu H, Fujiwara T (2007) Spectral fitting for signal assignment and structural analysis of uniformly 13C-labeled solid proteins by simulated annealing based on chemical shifts and spin dynamics. J Biomol NMR 38:325–339CrossRefGoogle Scholar
  41. Metropolis N, Rosenbluth AW, Rosenbluth MN, Teller AH, Teller E (1953) Equation of state calculations by fast computing machines. J Chem Phys 21:1087–1092ADSCrossRefGoogle Scholar
  42. Neal S, Nip AM, Zhang H, Wishart DS (2003) Rapid and accurate calculation of protein 1H, 13C and 15N chemical shifts. J Biomol NMR 26:215–240CrossRefGoogle Scholar
  43. Pikal MJ, Rigsbee DR, Roy ML (2007) Solid state chemistry of proteins: I. Glass transition behavior in freeze dried disaccharide formulations of human growth hormone (hGH). J Pharm Sci 96:2765–2776CrossRefGoogle Scholar
  44. Reckel S, Lopez JJ, Lohr F, Glaubitz C, Dotsch V (2012) In-cell solid-state NMR as a tool to study proteins in large complexes. ChemBioChem 13:534–537CrossRefGoogle Scholar
  45. Renault M, Tommassen-van Boxtel R, Bos MP, Post JA, Tommassen J, Baldus M (2012) Cellular solid-state nuclear magnetic resonance spectroscopy. Proc Natl Acad Sci USA 109:4863–4868ADSCrossRefGoogle Scholar
  46. Schubert M, Manolikas T, Rogowski M, Meier BH (2006) Solid-state NMR spectroscopy of 10% 13C labeled ubiquitin: spectral simplification and stereospecific assignment of isopropyl groups. J Biomol NMR 35:167–173CrossRefGoogle Scholar
  47. Schuetz A, Wasmer C, Habenstein B, Verel R, Greenwald J, Riek R, Bockmann A, Meier BH (2010) Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1–227). ChemBioChem 11:1543–1551CrossRefGoogle Scholar
  48. Seidel K, Etzkorn M, Heise H, Becker S, Baldus M (2005) High-resolution solid-state NMR studies on uniformly [13C,15N]-labeled ubiquitin. ChemBioChem 6:1638–1647CrossRefGoogle Scholar
  49. Seidel K, Etzkorn M, Schneider R, Ader C, Baldus M (2009) Comparative analysis of NMR chemical shift predictions for proteins in the solid phase. Solid State Nucl Magn Reson 35:235–242CrossRefGoogle Scholar
  50. Shen Y, Bax A (2007) Protein backbone chemical shifts predicted from searching a database for torsion angle and sequence homology. J Biomol NMR 38:289–302CrossRefGoogle Scholar
  51. Shen Y, Lange O, Delaglio F, Rossi P, Aramini JM, Liu G, Eletsky A, Wu Y, Singarapu KK, Lemak A, Ignatchenko A, Arrowsmith CH, Szyperski T, Montelione GT, Baker D, Bax A (2008) Consistent blind protein structure generation from NMR chemical shift data. Proc Natl Acad Sci USA 105:4685–4690ADSCrossRefGoogle Scholar
  52. Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS plus: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44:213–223CrossRefGoogle Scholar
  53. Simons KT, Kooperberg C, Huang E, Baker D (1997) Assembly of protein tertiary structures from fragments with similar local sequences using simulated annealing and Bayesian scoring functions. J Mol Biol 268:209–225CrossRefGoogle Scholar
  54. Steinbach PJ, Brooks BR (1993) Protein hydration elucidated by molecular-dynamics simulation. Proc Natl Acad Sci USA 90:9135–9139ADSCrossRefGoogle Scholar
  55. Takegoshi K, Nakamura S, Terao T (2001) C-13-H-1 dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem Phys Lett 344:631–637ADSCrossRefGoogle Scholar
  56. Todokoro Y, Yumen I, Fukushima K, Kang SW, Park JS, Kohno T, Wakamatsu K, Akutsu H, Fujiwara T (2006) Structure of tightly membrane-bound mastoparan-X, a G-protein-activating peptide, determined by solid-state NMR. Biophys J 91:1368–1379CrossRefGoogle Scholar
  57. Todokoro Y, Kobayashi M, Sato T, Kawakami T, Yumen I, Aimoto S, Fujiwara T, Akutsu H (2010) Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthase by solid-state NMR. J Biomol NMR 48:1–11CrossRefGoogle Scholar
  58. Van Melckebeke H, Wasmer C, Lange A, Ab E, Loquet A, Bockmann A, Meier BH (2010) Atomic-resolution three-dimensional structure of HET-s(218–289) amyloid fibrils by solid-state NMR spectroscopy. J Am Chem Soc 132:13765–13775CrossRefGoogle Scholar
  59. Vijay-Kumar S, Bugg CE, Cook WJ (1987) Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol 194:531–544CrossRefGoogle Scholar
  60. Wang G, Dunbrack RL Jr (2005) PISCES: recent improvements to a PDB sequence culling server. Nucleic Acids Res 33:W94–W98CrossRefGoogle Scholar
  61. Welsh LC, Symmons MF, Marvin DA (2000) The molecular structure and structural transition of the alpha-helical capsid in filamentous bacteriophage Pf1. Acta Crystallogr D Biol Crystallogr 56:137–150CrossRefGoogle Scholar
  62. Wishart DS, Sykes BD, Richards FM (1991) Relationship between nuclear magnetic resonance chemical shift and protein secondary structure. J Mol Biol 222:311–333CrossRefGoogle Scholar
  63. Wishart DS, Sykes BD, Richards FM (1992) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31:1647–1651CrossRefGoogle Scholar
  64. Wishart DS, Arndt D, Berjanskii M, Tang P, Zhou J, Lin G (2008) CS23D: a web server for rapid protein structure generation using NMR chemical shifts and sequence data. Nucleic Acids Res 36:W496–W502CrossRefGoogle Scholar
  65. Zech SG, Wand AJ, McDermott AE (2005) Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin. J Am Chem Soc 127:8618–8626CrossRefGoogle Scholar
  66. Zhou DH, Shea JJ, Nieuwkoop AJ, Franks WT, Wylie BJ, Mullen C, Sandoz D, Rienstra CM (2007) Solid-state protein-structure determination with proton-detected triple-resonance 3D magic-angle-spinning NMR spectroscopy. Angew Chem Int Ed 46:8380–8383CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2012

Authors and Affiliations

  • Keisuke Ikeda
    • 1
    • 2
  • Ayako Egawa
    • 1
  • Toshimichi Fujiwara
    • 1
  1. 1.Institute for Protein ResearchOsaka UniversitySuitaJapan
  2. 2.Graduate School of Medicine and Pharmaceutical SciencesUniversity of ToyamaToyamaJapan

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