Figure 3

The use of temperature resistant bicelles at lower temperatures enables the measurement of the transmembrane structure and topology of the functional form of microsomal cytochrome P450 2B4.

(A) A UV-Vis spectrum shows that the overall folding of the catalytic site of a heat-sensitive membrane protein, cytochrome P450 2B4, can be stabilized in the functional form using temperature resistant bicelles, composed of DLPC and DHPC. (B) One-dimensional cross-polarization experiments of 100 μl of magnetically-aligned DLPC/DHPC bicelles (q = [DLPC]/[DHPC] = 4.0) containing a 0.61 mM of a uniformly-¹⁵N-labeled cytochrome P450 2B4 shows anisotropic ¹⁵N chemical shifts at 15°C. (C) Two-dimensional HIMSELF experiment reveal that cytochrome P450 2B4 has a helical structure in the N-terminal transmembrane region. The amino acid sequence (D) and a helical wheel representation (E) of the N-terminal transmembrane region of cytochrome P450 2B4⁵⁴. The full length amino acid sequence is given in the Supporting Information. Hydrophobic and hydrophilic amino acids are in black and blue, respectively. (F) A model depicting the structure and topology of cytochrome P450 in lipid bilayers; the soluble domain structure is adapted from the crystal structure for amino acid residues 28–491^44,45,46. The transmembrane structure of residues 1–27, obtained using the structure assembly simulation, I-TASSER⁵⁵, is shown in the lipid bilayer region; the structure obtained with the highest C-score⁵⁵ was chosen. The transmembrane domain may not be a straight α-helix due to the presence of Gly residues. This result is consistent with the imperfect wheel-like pattern of resonances in the 2D HIMSELF spectrum shown in Figure 3(C).