Liquid crystalline phase of G-tetrad DNA for NMR study of detergent-solubilized proteins

Abstract

The liquid crystalline phase consisting of the potassium salt of the dinucleotide d(GpG) is compatible with detergents commonly used for solubilizing membrane proteins, including dodecylphosphocholine, the lysolipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, and small bicelles consisting of dihexanoyl phosphatidylcholine and dimyristoyl phosphatidylcholine. The chiral nematic liquid crystalline phase of d(GpG) consists of long columns of stacked G-tetrad structures and carry a net negative charge. For water-soluble systems, the protein alignment induced by d(GpG) is very similar to that observed for liquid crystalline Pf1 bacteriophage, but of opposite sign. Alignment of the detergent-solubilized fusion domain of hemagglutinin is demonstrated to be homogeneous and stable, resulting in high quality NMR spectra suitable for the measurement of residual dipolar couplings.

Figure 1

Residual ²H₂O quadrupole coupling in the presence of d(GpG). (A) Spectra of ²H₂O with and without DPC and (B) the dependence of the ²H₂O RQC on the concentration of d(GpG). (A) RQCs are 21.9 Hz (DPC, pH 5.8), 20.6 Hz (DPC, pH 6.6), 21.9 Hz (DPC, pH 7.4) and 25.0 Hz (pH7.4). The pH titration was carried out on a sample of 3 mM U-{¹³C,¹⁵N-gly,ala}-HAfp and 28 mg/ml d(GpG), 23 mM K₂HPO₄, 81 mM DPC on a Bruker 500 MHz DMX NMR spectrometer at 32 °C. See footnote to Table 1 for sample details on the sample without DPC. (B) Concentration dependence of the ²H₂O residual quadrupolar coupling.

Figure 2

¹H−¹⁵N IPAP-HSQC²⁰ and J_CαHα-coupled HNCA spectra for 2.1 mM U-{¹³C,¹⁵N-gly,ala}-HAfp [Biopeptide, sequence: GLFGAIAG FIENGWEG MIDG-OH] in 81 mM DPC [Anatrace], 23 mM K₂HPO₄, pH 7.4. J_NH and J_CH splittings are shown for the isotropic sample. RDCs (D_NH and D_CH), obtained from the change in splitting are shown for the spectra recorded after addition of 28 mg/ml d(GpG). Spectra containing the upfield (blue) and downfield (red) ¹⁵N-{¹H} doublet components of the ¹H−¹⁵N IPAP-HSQC spectra are superimposed. Measurements conducted on a Bruker 500 MHz DMX NMR spectrometer at 32 °C.