Comparison of alignment tensors generated for native tRNAVal using magnetic fields and liquid crystalline media
- 143 Downloads
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.
KeywordsAlignment 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.
- 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
- Bax A, Kontaxis G, Tjandra N (2001) Dipolar couplings in macromolecular structure determination. Methods Enzymol 339:127–174Google Scholar
- Bothner-By AA (1995) In: Grant DM, Harris RK (eds) Encyclopedia of nuclear magnetic resonance. Wiley, Chichester, pp 2932–2938Google Scholar
- Lilley DM (2004) Analysis of global conformational transitions in ribozymes. Methods Mol Biol 252:77–108Google Scholar
- 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
- 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
- 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
- Vermeulen A (2003) Determining nucleic acid global structure by application of NMR residual dipolar couplings. Dissertation, University of ColoradoGoogle Scholar
- Yue D (1994) Structure and function of unmodified E. coli valine-tRNA. Dissertation, Iowa State UniversityGoogle Scholar