title: DNA nanotubes for NMR structure determination of membrane proteins.
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.
authors.affiliation: 1] Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. [2] Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. [3] Wyss Institute for Biologically Inspired Engineering at Harvard, Boston, Massachusetts, USA.
authors.forename: Gaëtan
authors.initials: G
authors.lastname: Bellot
authors.forename: Mark A
authors.initials: MA
authors.lastname: McClintock
authors.forename: James J
authors.initials: JJ
authors.lastname: Chou
authors.forename: William M
authors.initials: WM
authors.lastname: Shih
mesh_terms: D004247:DNA / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D043942:Nanotubes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D008823:Micelles; D004247:DNA