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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.

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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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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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  1. Mark R. Hansen
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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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