The amphipathic helix 0 of endophilin (i.e., H0-Endo) is important to membrane binding, but its function of curvature generation remains controversial. We used electron paramagnetic resonance (EPR) spectroscopy to study effects of H0-Endo on membrane material properties. We found that H0-Endo reduced lipid chain mobility and increased bilayer polarity, i.e., making the bilayer interior more polar. Lipid-dependent examination revealed that anionic lipids augmented the effect of H0-Endo, while cholesterol had a minimal impact. Our EPR spectroscopy of magnetically aligned bicelles showed that as the peptide-to-lipid ratio increased, the lipid chain orientational order decreased gradually, followed by a sudden loss... More
The amphipathic helix 0 of endophilin (i.e., H0-Endo) is important to membrane binding, but its function of curvature generation remains controversial. We used electron paramagnetic resonance (EPR) spectroscopy to study effects of H0-Endo on membrane material properties. We found that H0-Endo reduced lipid chain mobility and increased bilayer polarity, i.e., making the bilayer interior more polar. Lipid-dependent examination revealed that anionic lipids augmented the effect of H0-Endo, while cholesterol had a minimal impact. Our EPR spectroscopy of magnetically aligned bicelles showed that as the peptide-to-lipid ratio increased, the lipid chain orientational order decreased gradually, followed by a sudden loss. We discuss an interfacial-bound model of the amphipathic H0-Endo to account for all EPR data. We used atomic force microscopy and fluorescence microscopy to explore membrane morphological changes. We found that H0-Endo caused the formation of micron-sized holes in mica-supported planar bilayers. Hole formation is likely caused by two competing forces - the adhesion force exerted by the substrate represses bilayer budging, whereas the line tension originating from peptide clustering has a tendency of destabilizing bilayer organization. In the absence of substrate influences, membrane curvature induction was manifested by generating small vesicles surrounding giant unilamellar vesicles. Our results of membrane perforation and vesiculation suggest that the functionality of H0-Endo is more than just coordinating membrane binding of endophilin.
