Journal of Biomolecular NMR

, Volume 40, Issue 2, pp 83–94 | Cite as

Comparison of alignment tensors generated for native tRNAVal using magnetic fields and liquid crystalline media

  • Michael P. Latham
  • Paul Hanson
  • Darin J. Brown
  • Arthur PardiEmail author


Residual dipolar couplings (RDCs) complement standard NOE distance and J-coupling torsion angle data to improve the local and global structure of biomolecules in solution. One powerful application of RDCs is for domain orientation studies, which are especially valuable for structural studies of nucleic acids, where the local structure of a double helix is readily modeled and the orientations of the helical domains can then be determined from RDC data. However, RDCs obtained from only one alignment media generally result in degenerate solutions for the orientation of multiple domains. In protein systems, different alignment media are typically used to eliminate this orientational degeneracy, where the combination of RDCs from two (or more) independent alignment tensors can be used to overcome this degeneracy. It is demonstrated here for native E. coli tRNAVal that many of the commonly used liquid crystalline alignment media result in very similar alignment tensors, which do not eliminate the 4-fold degeneracy for orienting the two helical domains in tRNA. The intrinsic magnetic susceptibility anisotropy (MSA) of the nucleobases in tRNAVal was also used to obtain RDCs for magnetic alignment at 800 and 900 MHz. While these RDCs yield a different alignment tensor, the specific orientation of this tensor combined with the high rhombicity for the tensors in the liquid crystalline media only eliminates two of the four degenerate orientations for tRNAVal. Simulations are used to show that, in optimal cases, the combination of RDCs obtained from liquid crystalline medium and MSA-induced alignment can be used to obtain a unique orientation for the two helical domains in tRNAVal.


Alignment tensor Liquid crystalline medium Magnetic susceptibility anisotropy RDC RNA Domain orientation 



We thank Gabe Gittings for purification of the fd and fd mutant bacteriophage, Dr. Jinfa Ying for advice in acquiring the NMR spectra for magnetic alignment, Dr. Alexander Grishaev for the FORTRAN program for calculating the MSA-induced alignment tensor and Dr. Ad Bax for critical advice in collection of the MSA-induced RDCs and for valuable discussions. This work is supported in part by NIH grant AI33098, and MPL was supported in part by a NIH training grant T32 GM65103. The NMR instrumentation was purchased with partial support from NIH grants RR11969, RR16649 and GM068928, NSF grants 9602941 and 0230966, and the W. M. Keck Foundation.

Supplementary material


  1. Al-Hashimi HM, Valafar H, Terrell M, Zartler ER, Eidsness MK, Prestegard JH (2000) Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings. J Magn Reson 143:402–406CrossRefADSGoogle Scholar
  2. Al-Hashimi HM, Majumdar A, Gorin A, Kettani A, Skripkin E, Patel DJ (2001) Field- and phage-induced dipolar couplings in a homodimeric DNA quadruplex, relative orientation of G·(C-A) triad and G-tetrad motifs and direct determination of C2 symmetry axis orientation. J Am Chem Soc 123:633–640CrossRefGoogle Scholar
  3. Amiri KMA, Hagerman PJ (1994) Global conformation of a self-cleaving hammerhead RNA. Biochemistry 33:13172–13177CrossRefGoogle Scholar
  4. Bax A (2003) Weak alignment offers new NMR opportunities to study protein structure and dynamics. Protein Sci 12:1–16CrossRefGoogle Scholar
  5. Bax A, Kontaxis G, Tjandra N (2001) Dipolar couplings in macromolecular structure determination. Methods Enzymol 339:127–174Google Scholar
  6. Bondensgaard K, Mollova ET, Pardi A (2002) The global conformation of the hammerhead ribozyme determined using residual dipolar couplings. Biochemistry 41:11532–11542CrossRefGoogle Scholar
  7. Bothner-By AA (1995) In: Grant DM, Harris RK (eds) Encyclopedia of nuclear magnetic resonance. Wiley, Chichester, pp 2932–2938Google Scholar
  8. Bruschweiler R, Liao XB, Wright PE (1995) Long-range motional restrictions in a multidomain zinc-finger protein from anisotropic tumbling. Science 268:886–889CrossRefADSGoogle Scholar
  9. Bryce DL, Boisbouvier J, Bax A (2004) Experimental and theoretical determination of nucleic acid magnetic susceptibility: importance for the study of dynamics by field-induced residual dipolar couplings. J Am Chem Soc 126:10820–10821CrossRefGoogle Scholar
  10. Clore GM, Starich MR, Gronenborn AM (1998) Measurement of residual dipolar couplings of macromolecules in the nematic phase of a colloidal suspension of rod-shaped viruses. J Am Chem Soc 120:10571–10572CrossRefGoogle Scholar
  11. Cordier F, Dingley AJ, Grzesiek S (1999) A doublet-separated sensitivity-enhanced HSQC for the determination of scalar and dipolar one-bond J-couplings. J Biomol NMR 13:175–180CrossRefGoogle Scholar
  12. Davis JH, Tonelli M, Scott LG, Jaeger L, Williamson JR, Butcher SE (2005) RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex. J Mol Biol 351:371–382CrossRefGoogle Scholar
  13. 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 6:277–293CrossRefGoogle Scholar
  14. Friederich MW, Hagerman PJ (1997) The angle between the anticodon and aminoacyl acceptor stems of yeast tRNA(Phe) is strongly modulated by magnesium ions. Biochemistry 36:6090–6099CrossRefGoogle Scholar
  15. Gayathri C, Bothner-By AA, Vanzijl PCM, Maclean C (1982) Dipolar magnetic-field effects in NMR-spectra of liquids. Chem Phys Lett 87:192–196CrossRefADSGoogle Scholar
  16. Getz M, Sun X, Casiano-Negroni A, Zhang Q, Al-Hashimi HM (2007) Review NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings. Biopolymers 86:384–402CrossRefGoogle Scholar
  17. Hansen MR, Mueller L, Pardi A (1998) Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Biol 5:1065–1074CrossRefGoogle Scholar
  18. Hansen MR, Hanson P, Pardi A (2000) Filamentous bacteriophage as a versatile method for aligning RNA, DNA and proteins for measurement of NMR dipolar coupling interactions. Methods Enzymol 317:220–240CrossRefGoogle Scholar
  19. Latham MP, Brown DJ, McCallum SA, Pardi A (2005) NMR methods for studying the structure and dynamics of RNA. Chembiochem 6:1492–1505CrossRefGoogle Scholar
  20. Lilley DM (2004) Analysis of global conformational transitions in ribozymes. Methods Mol Biol 252:77–108Google Scholar
  21. Lipsitz RS, Tjandra N (2004) Residual dipolar couplings in NMR structure analysis. Annu Rev Biophys Biomol Struct 33:387–413CrossRefGoogle Scholar
  22. Losonczi JA, Prestegard JH (1998) Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules. J Biomol NMR 12:447–451CrossRefGoogle Scholar
  23. Losonczi JA, Andrec M, Fischer MW, Prestegard JH (1999) Order matrix analysis of residual dipolar couplings using singular value decomposition. J Magn Reson 138:334–342CrossRefADSGoogle Scholar
  24. Lukavsky PJ, Kim I, Otto GA, Puglisi JD (2003) Structure of HCV IRES domain II determined by NMR. Nat Struct Biol 10:1033–1038CrossRefGoogle Scholar
  25. Mollova ET, Hansen MR, Pardi A (2000) Global structure of RNA determined with residual dipolar couplings. J Am Chem Soc 122:11561–11562CrossRefGoogle Scholar
  26. Padrta P, Stefl R, Kralik L, Zidek L, Sklenár V (2002) Refinement of d(GCGAAGC) hairpin structure using one- and two-bond residual dipolar couplings. J Biomol NMR 24:1–14CrossRefGoogle Scholar
  27. 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 94:12366–12371CrossRefADSGoogle Scholar
  28. Piotto M, Saudek V, Sklenár V (1992) Gradient-tailored excitation for single-quantum NMR-spectroscopy of aqueous-solutions. J Biomol NMR 2:661–665CrossRefGoogle Scholar
  29. Ramirez BE, Bax A (1998) Modulation of the alignment tensor of macromolecules dissolved in a dilute liquid crystalline medium. J Am Chem Soc 120:9106–9107CrossRefGoogle Scholar
  30. Richards RJ, Wu H, Trantirek L, O’Connor CM, Collins K, Feigon J (2006) Structural study of elements of tetrahymena telomerase RNA stem-loop IV domain important for function. RNA 12:1475–1485CrossRefGoogle Scholar
  31. Rodriguez-Castaneda F, Haberz P, Leonov A, Griesinger C (2006) Paramagnetic tagging of diamagnetic proteins for solution NMR. Magn Reson Chem 44 Spec No: S10–S16Google Scholar
  32. Ruckert M, Otting G (2000) Alignment of biological macromolecules in novel nonionic liquid crystalline media for NMR experiments. J Am Chem Soc 122:7793–7797CrossRefGoogle Scholar
  33. Sass J, Cordier F, Hoffmann A, Cousin A, Omichinski JG, Lowen H, Grzesiek S (1999) Purple membrane induced alignment of biological macromolecules in the magnetic field. J Am Chem Soc 121:2047–2055CrossRefGoogle Scholar
  34. Sass HJ, Musco G, Stahl SJ, Wingfield PT, Grzesiek S (2000) Solution NMR of proteins within polyacrylamide gels: diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes. J Biomol NMR 18:303–309CrossRefGoogle Scholar
  35. Staple DW, Butcher SE (2003) Solution structure of the HIV-1 frameshift inducing stem-loop RNA. Nucleic Acids Res 31:4326–4331CrossRefGoogle Scholar
  36. Tjandra N, Bax A (1997) Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278:1111–1114CrossRefADSGoogle Scholar
  37. 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 92:9279–9283CrossRefADSGoogle Scholar
  38. Tycko R, Blanco FJ, Ishii Y (2000) Alignment of biopolymers in strained gels: a new way to create detectable dipole-dipole couplings in high-resolution biomolecular NMR. J Am Chem Soc 122:9340–9341CrossRefGoogle Scholar
  39. Valafar H, Prestegard JH (2004) REDCAT: a residual dipolar coupling analysis tool. J Magn Reson 167:228–241CrossRefADSGoogle Scholar
  40. van Buuren BNM, Schleucher A, Wittmann V, Griesinger C, Schwalbe H, Wijmenga SS (2004) NMR spectroscopic determination of the solution structure of a branched nucleic acid from residual dipolar couplings by using isotopically labeled nucleotides. Angewandte Chemie-Int Ed 43:187–192CrossRefGoogle Scholar
  41. Vermeulen A (2003) Determining nucleic acid global structure by application of NMR residual dipolar couplings. Dissertation, University of ColoradoGoogle Scholar
  42. Vermeulen A, Zhou H, Pardi A (2000) Determining DNA global structure and DNA bending by application of NMR residual dipolar couplings. J Am Chem Soc 122:9638–9647CrossRefGoogle Scholar
  43. Vermeulen A, McCallum SA, Pardi A (2005) Comparison of the structure and dynamics of native and unmodified tRNAval. Biochemistry 44:6024–6033CrossRefGoogle Scholar
  44. Wu B, Petersen M, Girard F, Tessari M, Wijmenga SS (2006) Prediction of molecular alignment of nucleic acids in aligned media. J Biomol NMR 35:103–115CrossRefGoogle Scholar
  45. Ying J, Grishaev A, Latham MP, Pardi A, Bax A (2007) Magnetic field induced residual dipolar couplings of imino groups in nucleic acids from measurements at a single magnetic field. J Biomol NMR 39:91–96CrossRefGoogle Scholar
  46. Yue D (1994) Structure and function of unmodified E. coli valine-tRNA. Dissertation, Iowa State UniversityGoogle Scholar
  47. Zweckstetter M, Bax A (2000) Prediction of sterically induced alignment in a dilute liquid crystalline phase: aid to protein structure determination by NMR. J Am Chem Soc 122:3791–3792CrossRefGoogle Scholar
  48. Zweckstetter M, Bax A (2002) Evaluation of uncertainty in alignment tensors obtained from dipolar couplings. J Biomol NMR 23:127–137CrossRefGoogle Scholar
  49. Zweckstetter M, Hummer G, Bax A (2004) Prediction of charge-induced molecular alignment of biomolecules dissolved in dilute liquid-crystalline phases. Biophys J 86:3444–3460CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Michael P. Latham
    • 1
  • Paul Hanson
    • 1
  • Darin J. Brown
    • 1
    • 2
  • Arthur Pardi
    • 1
    Email author
  1. 1.Department of Chemistry and Biochemistry, 215 UCBUniversity of Colorado, BoulderBoulderUSA
  2. 2.Department of BiochemistryUniversity of Colorado at Denver, Health Science CenterAuroraUSA

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