DNA-nanotube-induced alignment of membrane proteins for NMR structure determination
- Shawn M. Douglas*,†,‡,
- James J. Chou*,§, and
- William M. Shih*,‡,§
- Departments of *Biological Chemistry and Molecular Pharmacology and
- †Genetics, Harvard Medical School, Boston, MA 02115; and
- ‡Department of Cancer Biology, Dana–Farber Cancer Institute, Boston, MA 02115
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Communicated by Tom A. Rapoport, Harvard Medical School, Boston, MA, January 31, 2007 (received for review December 15, 2006)
Abstract
Membrane proteins are encoded by 20–35% of genes but represent <1% of known protein structures to date. Thus, improved methods for membrane-protein structure determination are of critical importance. Residual dipolar couplings (RDCs), commonly measured for biological macromolecules weakly aligned by liquid-crystalline media, are important global angular restraints for NMR structure determination. For α-helical membrane proteins >15 kDa in size, Nuclear-Overhauser effect-derived distance restraints are difficult to obtain, and RDCs could serve as the main reliable source of NMR structural information. In many of these cases, RDCs would enable full structure determination that otherwise would be impossible. However, none of the existing liquid-crystalline media used to align water-soluble proteins are compatible with the detergents required to solubilize membrane proteins. We report the design and construction of a detergent-resistant liquid crystal of 0.8-μm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4-μm-long six-helix bundles each self-assembled from a 7.3-kb scaffold strand and >170 short oligonucleotide staple strands. We show that the DNA-nanotube liquid crystal enables the accurate measurement of backbone NH and CαHα RDCs for the detergent-reconstituted ζ-ζ transmembrane domain of the T cell receptor. The measured RDCs validate the high-resolution structure of this transmembrane dimer. We anticipate that this medium will extend the advantages of weak alignment to NMR structure determination of a broad range of detergent-solubilized membrane proteins.
Footnotes
- §To whom correspondence may be addressed. E-mail: william_shih{at}dfci.harvard.edu or james_chou{at}hms.harvard.edu
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Author contributions: S.M.D., J.J.C., and W.M.S. designed research, performed research, analyzed data, and wrote the paper.
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Conflict of interest statement: S.M.D. and W.M.S. declare competing financial interests. A provisional patent entitled “Nucleic-acid-nanotube liquid crystals and use for NMR structure determination of detergent-solubilized membrane proteins” was filed on April 21, 2006 on behalf of the Dana–Farber Cancer Institute by Edwards Angell Palmer & Dodge LLP, listing S.M.D. and W.M.S. as coinventors. A planned update to the patent filing will add J.J.C. of Harvard Medical School as a coinventor.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0700930104/DC1.
- Abbreviations:
- RDC,
- residual dipolar coupling;
- SVD,
- singular value decomposition.
- © 2007 by The National Academy of Sciences of the USA
