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Research Themes Membrane proteins

Membrane Proteome: Making DNA Nanotubes for NMR Structure Determination

SBKB [doi:10.1038/sbkb.2012.155]
Technical Highlight - August 2013
Short description: A protocol for designing DNA nanotube liquid crystals to be used as a weak-alignment medium for high-resolution NMR is presented.

Schematic view of the folded six-helix bundle DNA nanotube, composed of scaffold and staples; a cross-sectional view is shown at the bottom. 1

The structural elucidation of membrane proteins via solution NMR presents particular challenges, given the α-helical nature of transmembrane segments and the requirement for solubilizing detergents. Assigning side-chain methyl resonances is extremely difficult, due to the added molecular weight of detergent micelles and limited chemical-shift dispersion of high methyl-bearing residue content in α-helical segments, resulting in resonance overlap. The weak alignment of proteins, aided by large molecules that form liquid crystals at low concentration, can provide global orientation restraints that greatly facilitate NMR structure determination. However, previously reported weak alignment media are not compatible with detergents; for example, Pf1 bacteriophage is a popular alignment medium, although it denatures in the presence of detergents required to solubilize membrane proteins.

To overcome these limitations, Shih and colleagues have developed DNA nanotube liquid crystals as an alternative weak-alignment medium that is generally suitable for high-resolution NMR structure determination. The chemical properties of DNA allow for the weak alignment of membrane proteins over a wide selection of detergents, pH and temperatures, making DNA nanotube-based media especially attractive in the study of membrane proteins. The generation of this versatile medium for NMR is now made accessible by the publication of a detailed step-by-step protocol.

Although a comprehensive understanding of DNA nanotube design is not required to successfully generate useful media, the protocol offers a complete description of the original design that allows user adjustments to potentially suit any purpose. In their basic iteration, nanotubes suitable for NMR applications extend to a uniform length of 0.8 μM by linking two 0.4 μM-monomers assembled separately using the DNA origami method. To assemble one monomer, an M13 phage-derived, 7,308-nucleotide, single-stranded circular DNA is used as a 'scaffold.' This substrate is then folded by the addition of excess 'staples': single-stranded oligonucleotides, 42 nt in length, that contain three 14-nt regions of discontinuous complementarity to the scaffold, resulting in the assembly of six parallel double helices curled into a tube. The design is such that separately assembled 'front' and 'rear' monomers can be combined to form dimers through complementary unpaired staple strands. The purified dimers are then concentrated to ∼25 mg ml−1, a concentration at which they spontaneously align to form a stable liquid crystal.

The protocol also offers suggestions to adapt the method for problematic samples, such as proteins with a high net positive charge whose interaction with DNA can reduce the tumbling rate and impact the accuracy of residual dipolar coupling measurements. A single individual should be able to produce sufficient nanotubes for up to five NMR experiments within one week.

Stéphane Larochelle

References

  1. G. Bellot et al. DNA nanotubes for NMR structure determination of membrane proteins.
    Nat Protoc. 8, 755-770 (2013). doi:10.1038/nprot.2013.037

  2. S.M. Douglas, J.J. Chou and W.M. Shih. DNA-nanotube-induced alignment of membrane proteins for NMR structure determination.
    PNAS. 104, 6644-6648 (2007). doi:10.1073/pnas.0700930104

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