DNA-nanotube-induced alignment of membrane proteins for NMR structure determination

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 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.

Fig. 1.

DNA-nanotube design. (a) 3D cartoon view. The front and rear monomers are folded separately and then combined to form heterodimers. (b) Segment diagram. Each monomer consists of 28 segments of 42-bp length as well as a head and tail segment on each end. (c) Scaffold-only schematic view. Each monomer consists of a modified M13-bacteriophage single-stranded DNA genome of 7,308 bases in length serving as a “scaffold” and 168 DNA strands of 42-bp length acting as “staples” that constrict the scaffold to the target structure. (d) Cross-sectional view. (e) Scaffold-plus-staples schematic view of the heterodimer junction. There are two 42-bp staple strands (orange) per 14-bp subsegment. (f) Scaffold-plus-staples schematic view of a typical 42-bp segment. The crossover pattern of six staple strands repeats for every 42-bp segment along the length of the nanotube.

Fig. 2.

Characterization of DNA-nanotubes. (a) Gel-shift analysis of folding and heterodimerization of DNA-nanotubes. Lane M, marker lane with DNA size standards (number of base pairs indicated at the left); lane 1, M13-derived (p7308) ssDNA scaffold; lanes 2 and 3, front and rear folded monomers (scaffold plus staples); lanes 4 and 5, front and rear monomers after PEG fractionation; lane 6, heterodimers, after 2-h incubation of mixed monomers at 37°C. Samples were electrophoresed in a 2% agarose gel containing 11 mM MgCl₂, 0.5 μg·ml⁻¹ ethidium bromide, 45 mM Tris base, 45 mM boric acid, and 1 mM EDTA (pH 8.0). (b) Negative-stain electron micrographs of DNA-nanotube heterodimers. (Scale bar, 500 nm.) Samples were stained with 0.7% uranyl formate and imaged on a Tecnai G2 Spirit BioTWIN microscope. (c) Photograph of birefringence exhibited between crossed polarizers by DNA-nanotube dimers at 28 mg·ml⁻¹ in a glass NMR tube. (d) D ²H spectrum of a 10 mM NaH₂PO₄, 10 mM MgCl₂, 90% H₂O/10% D₂O, 100 mM DPC sample containing 28 mg·ml⁻¹ DNA-nanotube heterodimers. The liquid crystal was aligned in an 11.4 T magnetic field and yielded ²H quadrupolar splitting of 5.56 Hz.

Fig. 3.

Analysis of RDCs measured for detergent-reconstituted transmembrane domain of the ζ chain (residues 7–39) of the T cell receptor complex, weakly aligned in 28 mg·ml⁻¹ DNA-nanotube. (a) Correlation between observed backbone RDCs (normalized to D_NH) and RDCs predicted for the known NMR structure of ζ-ζ TM domain (PDB ID code 2HAC) by using an alignment tensor obtained from the SVD fit. The correlation coefficient R_SVD is 0.98, and Q_free is 16%. (b) Principal axes of the alignment tensor relative to 2HAC: A_zz, −19.8 Hz; A_yy, 15.2 Hz; A_xx, 4.6 Hz; D_aNH, −9.9 Hz; and rhombicity R, 0.357. The solid line in the ribbon structure of the ζ-ζ TM domain represents the axis of C₂ rotational symmetry.