In vitro membrane-inserted conformation of the cytochrome b5 tail

Abstract

The cytochrome b5 tail is a 43-residue membrane-embedded domain that is responsible for anchoring the catalytic domain of cytochrome b5 to the endoplasmic reticulum membrane. Different models for the structure of the membrane domain of cytochrome b5 have been proposed, including a helical hairpin and a single transmembrane helix. In the present study, CD spectroscopy was used to investigate the conformation of the tail in different environments, and as a function of temperature, with the goal of understanding the nature of the membrane-bound conformation. Whereas residue property profiling indicates that bending of a helix in the middle of the peptide might be possible, the experimental results in small unilamellar vesicles and lysophosphatidylcholine micelles are more consistent with a single transmembrane helix. Furthermore, although there is evidence for some refolding of the polypeptide with temperature, this is not consistent with a hairpin-to-transmembrane transition. Rather, it appears to represent an increase in helical content in fluid lipid environments, perhaps involving residues at the ends of the transmembrane segment.

Allosteric phenomena in cytochrome P450‐catalyzed monooxygenations

“…There is also compelling evidence of a functional interplay of the components in the b 5 /lipid couple: EPR measurements with artificial vesicles suggested motional restriction of phospholipid molecules adjacent to the hydrophobic segment of b 5 [149], the nature of the vesicle preparations used to reconstitute b 5 being a determinant of the structural orientation within the phospholipid bilayer of the hemoprotein's nonpolar tail [150]; the latter incurs a conformational change, when inserted in the tight configuration, to exhibit a more α helical overall structure as compared with the loose form [151]. These findings, together with the fact that the C‐terminal membrane anchor of b 5 plays a vital role in docking of the pigment to P450s [152–154], provide a rationale for the observed modulation by lipid of the apparent dissociation constant for complex formation between b 5 and CYP2B4 [155].…”

A nanoscaled Au–horseradish peroxidase composite fabricated by an interface reaction and its characterization, immobilization and biosensing

“…The CD spectrum of HRP exhibits a negative peak at ca.185 nm (curve a), which is similar to those of the other heme containing proteins. 37 The position of Au-HRP (curve b) is almost the same as that from HRP. The similarities between the CD spectra of curves a and b in Fig.…”

Inter-organelle interactions between the ER and mitotic spindle facilitates Zika protease cleavage of human Kinesin-5 and results in mitotic defects

“…First, we generated a mScarlet-labeled cytochrome b5 reductase chimera. Cytochrome b5 reductase is a well-characterized resident ER integral membrane protein (Borgese et al, 1993;Mitoma and Ito, 1992;Hanlon et al, 2000). Comprised of an exposed cytoplasmic catalytic domain and a transmembrane ER anchor helix (b5 anchor ), we substituted mScarlet (Bindels et al, 2017) for the catalytic domain of the reductase to generate an ER fluorescentmarker expression-construct in pcDNA3.1 (Figure S7).…”