DNA-nanotube-induced alignment of membrane proteins for NMR structure determination.
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 alpha-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-microm-long DNA-nanotubes that can be used to induce weak alignment of membrane proteins. The nanotubes are heterodimers of 0.4-microm-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 N(H) and C(alpha)H(alpha) RDCs for the detergent-reconstituted zeta-zeta 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.
Douglas
Shawn M
SM
Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115, USA.
Chou
James J
JJ
Shih
William M
WM
D017104:Bacteriophage M13 / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry*; D050866:Liquid Crystals; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry*; D036103:Nanotechnology; D043942:Nanotubes / Q000737:chemistry*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain