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. 2007 Jul 4;129(26):8078-9.
doi: 10.1021/ja0728371. Epub 2007 Jun 13.

Structural Similarity of a Membrane Protein in Micelles and Membranes

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Free PMC article

Structural Similarity of a Membrane Protein in Micelles and Membranes

Carla M Franzin et al. J Am Chem Soc. .
Free PMC article

Abstract

The anisotropic spin interactions measured for membrane proteins in weakly oriented micelles and in oriented lipid bilayers provide independent and potentially complementary high-resolution restraints for structure determination. Here we show that the membrane protein CHIF adopts a similar structure in lipid micelles and bilayers, allowing the restraints from micelle and bilayer samples to be combined in a complementary fashion to enhance the structural information. Back-calculation and assignment of the NMR spectrum of CHIF in oriented lipid bilayers, from the structure determined in micelles, provides additional restraints for structure determination as well as the global orientation of the protein in the membrane. The combined use of solution and solid-state NMR restraints also affords cross-validation for the structural analysis.

Figures

Figure 1
Figure 1
(A) Amino acid sequence of CHIF (NCBI accession: NP_068702). Residue numbering begins at 1 after the signal sequence. Leu in H1 and H2 are in red. Leu22 and Leu35 were introduced by mutations of native Met. (B) Secondary structure, 1H-15N RDCs, and 1H/15N heteronuclear NOEs (gray bars), for CHIF in micelles. Positions left blank correspond to Pro (3, 9, 49, 53, 58, 62) or overlapped peaks. Sample preparation and NMR experiments were as described, .
Figure 2
Figure 2
NMR spectra of 15N-labeled CHIF in lipid bilayers (B–G) oriented with the bilayer surface perpendicular to the magnetic field or (A) unoriented. (C) Sample exposed to D2O. (D–G) PISEMA spectra of the transmembrane region for (D) uniformly 15N-labeled or (E–G) Leu 15N-labeled CHIF. The dotted lines trace the wheel-like spectra calculated for ideal α-helices with different tilts. The red circles reproduce the experimental Leu spectrum. (E) The Leu spectrum (filled red circles) was calculated for two separate ideal α-helices with 15° (black line) or 20° (red line) tilt. Asterisks mark peaks that are expected to deviate from the wheel. (G) Leu spectrum back-calculated from the structure of CHIF after refinement with the dipolar couplings assigned in (F). Spectra were calculated with tensor values and FORTRAN code as described–. Sample preparation and NMR experiments were as described. Samples contained 2–4 mg of 15N-labeled protein, 80 mg of di-oleoyl-phosphatidyl-choline, and 20 mg of di-oleoyl-phosphatidyl-glycerol.
Figure 3
Figure 3
Structure of CHIF determined in micelles and oriented in the membrane using the data from oriented lipid bilayers. The structure in micelles (PDB code: 2JP3) was determined as described for its homolog FXYD1. Z is the axis perpendicular to the membrane surface.

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