Bilayer structure and physical dynamics of the cytochrome b5 dimyristoylphosphatidylcholine interaction

Abstract

Cytochrome b5 is a microsomal membrane protein which provides reducing potential to delta 5-, delta 6-, and delta 9-fatty acid desaturases through its interaction with cytochrome b5 reductase. Low angle x-ray diffraction has been used to determine the structure of an asymmetrically reconstituted cytochrome b5:DMPC model membrane system. Differential scanning calorimetry and fluorescence anisotropy studies were performed to examine the bilayer physical dynamics of this reconstituted system. These latter studies allow us to constrain structural models to those which are consistent with physical dynamics data. Additionally, because the nonpolar peptide secondary structure remains unclear, we tested the sensitivity of our model to different nonpolar peptide domain configurations. In this modeling approach, the nonpolar peptide moiety was arranged in the membrane to meet such chemically determined criteria as protease susceptibility of carboxyl- and amino-termini, tyrosine availability for pH titration and tryptophan 109 location, et cetera. In these studies, we have obtained a reconstituted cytochrome b5:DMPC bilayer structure at approximately 6.3 A resolution and conclude that the nonpolar peptide does not penetrate beyond the bilayer midplane. Structural correlations with calorimetry, fluorescence anisotropy and acyl chain packing data suggest that asymmetric cytochrome b5 incorporation into the bilayer increases acyl chain order. Additionally, we suggest that the heme peptide:bilayer interaction facilitates a discreet heme peptide orientation which would be dependent upon phospholipid headgroup composition.

In vitro membrane-inserted conformation of the cytochrome b5 tail

“…This is consistent with earlier neutron diffraction studies [59], which indicated that the tail penetrates deep into the lipid bilayer, but is at odds with low-angle X-ray scattering studies [60], which suggested that the tail only spans one-half of the bilayer (as it would in a hairpin). Fluorescence energy transfer measurements using modified lipids containing 9-anthroyloxy fatty acid probes showed that most of the fluorescence was associated with one of the three conserved Trp residues and that this residue was buried approximately 20 A / from the membrane surface in dimyristoyl phosphatidylcholine vesicles [61], a result that would be compatible with either a transmembrane structure or a hairpin.…”

α-Bungarotoxin Binding to Acetylcholine Receptor Membranes Studied by Low Angle X-Ray Diffraction

“…Disorder of the second kind, or lattice disorder, reflects the number of ordered membranes within a crystallite (Hosemann and Bagchi, 1962;Schwartz et al, 1975;Blaurock, 1982) and is a primary determinant of resolution. For our nAChR membrane multilayers, Patterson analysis (Chester et al, 1992;Young et al, 1992) revealed an average of three ordered membranes per crystallite (data not shown) with a mosaic spread of 30-408 ( Fig. 2 b).…”

“…Even with the apparent lattice disorder, lamellar diffraction was readily observed beyond 14 Å with a maximum Bragg-order of l ¼ 32 used in these studies (d/32 ¼ 14 Å , where d ¼ 452 Å ). This is not unexpected because reflections l ¼ 8, 16, and 32 arise primarily from the membrane lipid bilayer (Chester et al, 1992).…”

“…Modeling of the interaction between the nAChR and a-toxin was undertaken (Chester et al, 1992). For a careful analysis of the expected difference electron density profiles, the AChBP (1I9B; Brejc et al, 2001) and a-toxin (1KL8; Moise et al, 2002) were used in model calculations.…”

“…For a careful analysis of the expected difference electron density profiles, the AChBP (1I9B; Brejc et al, 2001) and a-toxin (1KL8; Moise et al, 2002) were used in model calculations. A complex of AChBP and two a-toxin molecules was constructed (Moise et al, 2002) and the number of electrons was projected onto the z axis parallel with the AChBP channel (Chester et al, 1992). The projected electron density profiles are atomic resolution models of the nAChR synaptic domain in the absence and presence of two a-toxin molecules.…”