Abstract
Finding a way to determine the structures of integral membrane proteins using solution nuclear magnetic resonance (NMR) spectroscopy has proved to be challenging. A residual-dipolar-coupling–based refinement approach can be used to resolve the structure of membrane proteins up to 40 kDa in size, but to do this you need a weak-alignment medium that is detergent-resistant and it has thus far been difficult to obtain such a medium suitable for weak alignment of membrane proteins. We describe here a protocol for robust, large-scale synthesis of detergent-resistant DNA nanotubes that can be assembled into dilute liquid crystals for application as weak-alignment media in solution NMR structure determination of membrane proteins in detergent micelles. The DNA nanotubes are heterodimers of 400-nm-long six-helix bundles, each self-assembled from a M13-based p7308 scaffold strand and >170 short oligonucleotide staple strands. Compatibility with proteins bearing considerable positive charge as well as modulation of molecular alignment, toward collection of linearly independent restraints, can be introduced by reducing the negative charge of DNA nanotubes using counter ions and small DNA-binding molecules. This detergent-resistant liquid-crystal medium offers a number of properties conducive for membrane protein alignment, including high-yield production, thermal stability, buffer compatibility and structural programmability. Production of sufficient nanotubes for four or five NMR experiments can be completed in 1 week by a single individual.
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Acknowledgements
We thank I. Ayala and J. Boisbouvier (CNRS, Institut de Biologie Structurale) for the gift of the ubiquitin vector and R. Sounier (Harvard Medical School) for helpful discussions. This work was supported by the US National Institutes of Health (NIH) grants 1U54GM094608 to J.J.C., and 1DP2OD004641 and 1U54GM094608 to W.M.S.
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B.G. and M.A.M. used, developed and troubleshot the technique. B.G. optimized procedures for NMR experiments, data processing and analysis. W.M.S. supervised the projects and J.J.C. participated in technology design and discussions. B.G. and W.M.S. wrote the manuscript, and J.J.C and M.A.M. revised the paper.
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J.J.C. and W.M.S. declare competing financial interests. A patent (US Patent application no. 13/090892) entitled “Nucleic-acid-nanotube liquid crystals and use for NMR structure determination of detergent-solubilized membrane proteins” was filed in 2007 on behalf of the Dana-Farber Cancer Institute and Harvard Medical School by Edwards Angell Palmer & Dodge LLP, listing J.J.C. and W.M.S. as two of the co-inventors.
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DNA oligonucleotide staple sequences (PDF 436 kb)
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Bellot, G., McClintock, M., Chou, J. et al. DNA nanotubes for NMR structure determination of membrane proteins. Nat Protoc 8, 755–770 (2013). https://doi.org/10.1038/nprot.2013.037
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DOI: https://doi.org/10.1038/nprot.2013.037
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