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Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions

Nature Structural Biology volume 5, pages 1065–1074 (1998)Cite this article

Abstract

Dipolar coupling interactions represent an extremely valuable source of long-range distance and angle information that was previously not available for solution structure determinations of macromolecules. This is because observation of these dipolar coupling data requires creating an anisotropic environment for the macromolecule. Here we introduce a new method for generating tunable degrees of alignment of macromolecules by addition of magnetically aligned Pf1 filamentous bacteriophage as a cosolute. This phage-induced alignment technique has been used to study 1H-1H, 1H-13C, and 1H-15N dipolar coupling interactions in a DNA duplex, an RNA hairpin and several proteins including thioredoxin and apo-calmodulin. The phage allow alignment of macromolecules over a wide range of temperature and solution conditions and thus represent a stable versatile method for generating partially aligned macromolecules in solution.

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Figure 1: 1D 2H spectra of a 10 mM TRIS (d11) pH 8.0, 90%/10% D2O sample containing 0, 9, 17, 29, 41 and 58 mg ml–1 Pf1 phage (top to bottom).
Figure 2: a, Sequence and secondary structures of the two IRE RNA hairpins studied here16.
Figure 3: Plot of the T1ρ 15N relaxation data for the G22 imino nitrogen in the 15N-labeled IRE-I RNA with and without 17 mg ml–1 Pf1 phage.
Figure 4: A portion of the 2D (15N,1H) HSQC spectra collected on bovine apo-calmodulin with no 1H decoupling in the t1 evolution period a, with no phage and b, with 25 mg ml–1 Pf1 phage.
Figure 5: The aromatic/amide proton regions of 1H-1H DQF-COSY spectra of thioredoxin a, with no phage b, with 17 mg ml–1 Pf1 phage.
Figure 6: a, The H1' to aromatic proton region of a 1H-1H DQF-COSY spectrum of the 16-mer DNA duplex dissolved in 20 mg ml–1 Pf1 phage.
Figure 7: Structure of part of the 16-mer DNA duplex illustrating the angles and distances for the interresidue and intraresidue H1' to H8/H6 connectivities.

References

  1. Prestegard, J.H. New techniques in structural NMR — anisotropic interactions. Nature Struct. Biol. 5, 517–522 (1998).

    Article  CAS  Google Scholar 

  2. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 ( 1997).

    Article  CAS  Google Scholar 

  3. Bothner-By, A.A. Magnetic field induced alignment of molecules. In Encyclopedia of nuclear magnetic resonance (eds Grant, D.M. & Harris, R.K.) 2932– 2938 (Wiley, Chichester; 1995).

    Google Scholar 

  4. Tjandra, N., Omichinski, J.G., Gronenborn, A.M., Clore, G.M. & Bax, A. Use of dipolar 1H-15N and 1H-13C couplings in the structure determination of magnetically oriented macromolecules in solution. Nature Struct. Biol. 4, 732–738 (1997).

    Article  CAS  Google Scholar 

  5. Tolman, J.R., Flanagan, J.M., Kennedy, M.A. & Prestegard, J.H. Nuclear magnetic dipole interactions in field-oriented proteins: information for structure determination in solution. Proc. Natl. Acad. Sci. USA 92, 9279–9283 ( 1995).

    Article  CAS  Google Scholar 

  6. Metz, G. et al. NMR studies of ubiquinone location in oriented model membranes—evidence for a single motionally averaged population. J. Am. Chem. Soc. 117, 564–565 ( 1995).

    Article  CAS  Google Scholar 

  7. Losonczi, J.A. & Prestegard, J.H. Nuclear magnetic resonance characterization of the myristoylated, N-terminal fragment of ADP-ribosylation factor 1 in a magnetically oriented membrane array. Biochemistry 37, 706–716 ( 1998).

    Article  CAS  Google Scholar 

  8. Torbet, J. & Maret, G. Fibres of highly oriented Pf1 bacteriophage produced in a strong magnetic field. J. Mol. Biol. 134, 843–845 (1979).

    Article  CAS  Google Scholar 

  9. Hill, D.F., Short, N.J., Perham, R.N. & Petersen, G.B. DNA sequence of the filamentous bacteriophage Pf1. J. Mol. Biol. 218, 349–364 ( 1991).

    Article  CAS  Google Scholar 

  10. Nambudripad, R., Stark, W. & Makowski, L. Neutron diffraction studies of the structure of filamentous bacteriophage Pf1. Demonstration that the coat protein consists of a pair of alpha-helices with an intervening, non-helical surface loop. J. Mol. Biol. 220, 359–379 (1991).

    Article  CAS  Google Scholar 

  11. Marvin, D.A. Filamentous phage structure, infection and assembly. Curr. Opin. Struct. Biol. 8, 150–158 (1998).

    Article  CAS  Google Scholar 

  12. Zimmermann, K., Hagedorn, H., Heuck, C.C., Hinrichsen, M. & Ludwig, H. The ionic properties of the filamentous bacteriophages Pf1 and fd. J. Biol. Chem. 261, 1653–1655 (1986).

    CAS  PubMed  Google Scholar 

  13. Bolon, P.J. & Prestegard, J.H. COSY cross-peaks from 1H-1H dipolar couplings in NMR spectra of field oriented oligosaccharides. J. Am. Chem. Soc. 120, 9366–9367 (1998).

    Article  CAS  Google Scholar 

  14. Hansen, M.R., Rance, M. & Pardi, A. Observation of long-range 1H-1H distances in solution by dipolar coupling interactions. J. Am. Chem. Soc. 120, 11210–11211 (1998).

    Article  CAS  Google Scholar 

  15. Tsang, P. & Opella, S.J. Pf1 virus particle dynamics. Biopolymers 25, 1859–1864 (1986).

    Article  CAS  Google Scholar 

  16. Addess, K.J., Basilion, J.P., Klausner, R.D., Rouault, T.A. & Pardi, A. Structure and dynamics of the iron responsive element RNA: implications for binding of the RNA by iron regulatory binding proteins. J. Mol. Biol. 274, 72– 83 (1997).

    Article  CAS  Google Scholar 

  17. Farrow, N.A., Zhang, O.W., Forman-Kay, J.D. & Kay, L.E. Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer. Biochemistry 34, 868–878 (1995).

    Article  CAS  Google Scholar 

  18. Tjandra, N. & Bax, A. Measurement of dipolar contributions to 1JCH splittings from magnetic-field dependence of J modulation in two-dimensional NMR spectra. J. Magn. Reson. 124, 512–515 ( 1997).

    Article  CAS  Google Scholar 

  19. Urbauer, J.L., Short, J.H., Dow, L.K. & Wand, A.J. Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium. Biochemistry 34, 8099–8109 (1995).

    Article  CAS  Google Scholar 

  20. Beger, R.D., Marathias, V.M., Volkman, B.F. & Bolton, P.H. Determination of internuclear angles of DNA using paramagnetic assisted magnetic alignment. J. Magn. Reson. 135, 256– 259 (1998).

    Article  CAS  Google Scholar 

  21. Bax, A. & Tjandra, N. High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid crystalline medium. J. Biomol. NMR 10, 289–292 (1997).

    Article  CAS  Google Scholar 

  22. Sanders, C.R.D. & Schwonek, J.P. Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solid-state NMR. Biochemistry 31, 8898–8905 ( 1992).

    Article  CAS  Google Scholar 

  23. Cross, T.A. & Opella, S.J. Hydrogen-1 and carbon-13 nuclear magnetic resonance of the aromatic residues of fd coat protein. Biochemistry 20, 290–297 (1981).

    Article  CAS  Google Scholar 

  24. Cross, T.A., Tsang, P. & Opella, S.J. Comparison of protein and deoxyribonucleic acid backbone structures in fd and Pf1 bacteriophages. Biochemistry 22, 721–726 (1983).

    Article  CAS  Google Scholar 

  25. Sanders, C.R., Hare, B.J., Howard, K.P. & Prestegard, J.H. Magnetically oriented phospholipid micelles as a tool for the study of membrane-associated molecules. Prog. Nucl. Magn. Resonance Spectrosc. 26 , 5 (1994).

    Article  Google Scholar 

  26. Clore, G.M., Gronenborn, A.M. & Tjandra, N. Direct structure refinement against residual dipolar couplings in the presence of rhombicity of unknown magnitude. J. Magn. Reson. 131, 159–162 (1998).

    Article  CAS  Google Scholar 

  27. Clore, G.M., Gronenborn, A.M. & Bax, A. A robust method for determining the magnitude of the fully asymmetric alignment tensor of oriented macromolecules in the absence of structural information. J. Magn. Reson. 133, 216–221 (1998).

    Article  CAS  Google Scholar 

  28. Clore, G.M., Starich, M.R. & Gronenborn A.M. 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, 10571–10572 ( 1998).

    Article  CAS  Google Scholar 

  29. Sambrook, J., Fritsch, E. & Maniatis, T. Molecular Cloning, a Laboratory Manual, 2.73–72.75 (Cold Spring Harbor Laboratory Press, Plainview, New York; 1989).

    Google Scholar 

  30. Kostrikis, L.G., Liu, D.J. & Day, L.A. Ultraviolet absorbance and circular dichroism of Pf1 virus: nucleotide/subunit ratio of unity, hyperchromic tyrosines and DNA bases, and high helicity in the subunits. Biochemistry 33, 1694–1703 (1994).

    Article  CAS  Google Scholar 

  31. Grzesiek, S. & Bax, A. The importance of not saturating H 2O in protein NMR—application to sensitivity enhancement and NOE measurements. J. Am. Chem. Soc. 115, 12593 –12594 (1993).

    Article  CAS  Google Scholar 

  32. Marion, D., Ikura, M., Tschudin, R. & Bax, A. Rapid recording of 2D NMR spectra without phase cycling - application to the study of hydrogen-exchange in proteins. J. Magn. Reson. 85, 393– 399 (1989).

    CAS  Google Scholar 

  33. Mattiello, D.L., Warren, W.S., Mueller, L. & Farmer, B.T. Minimizing the water resonance in biological NMR — characterization and suppression of intermolecular dipolar interactions by multiple-axis gradients. J. Am. Chem. Soc. 118, 3253– 3261 (1996).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from NIH (to A.P.) and a postdoctoral fellowship from the Leukemia Society of America (to M.R.H.). We thank A. Bax for providing the alignment tensor program, M.S. Friedrichs for modifications of the program, J. Wank for technical assistance, K.J. Addess and A.J. Wand for preparation of samples and M. Rance and D.S. Wuttke for valuable discussions.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, 80309-0215, Colorado, USA

    Mark R. Hansen & Arthur Pardi

  2. Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, 08543-4000, New Jersey , USA

    Luciano Mueller

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Correspondence to Arthur Pardi.

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Hansen, M., Mueller, L. & Pardi, A. Tunable alignment of macromolecules by filamentous phage yields dipolar coupling interactions. Nat Struct Mol Biol 5, 1065–1074 (1998). https://doi.org/10.1038/4176

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