Strikingly, we failed to identify a significant deviation of recombinant K35N N-BAR from WT in in vitro assays. This implies that the K35N mutation does not disrupt N-terminal helix hydrophobic insertion into the lipid membrane, which is a widely accepted mechanism for the N-BAR domain protein in curvature generation. Lastly,Danshensu we provided preliminary evidence that the cytoskeleton regulates membrane tubulation activity of BIN1 in cells. To further characterize the BIN1 N-BAR domain and its mutants, we purified WT BIN1 N-BAR domains as well as disease mutants. Because we neither observed retention volume shifts in size exclusion chromatography, nor any changes in CD spectra, we believe that none of the disease mutations had significant effects on the folding of the protein. To characterize curvature generation by BIN1 N-BAR domains and its mutants, we adopted an in vitro liposome deformation assay involving negative staining transmission electron microscopy. Negative staining is a widely accepted method to study biological macromolecules. It involves embedding liposomeprotein complexes adsorbed to sample grids in a dried heavy metal solution for contrast enhancement. We first used large unilamellar liposomes composed of Tanshinone-I DOPS to maximize electrostatic interactions between protein and membrane without the potential complications of lipid demixing. Contrary to the observations from cellular studies described above, tubules were found in all the samples when BIN1 variants were incubated with liposomes composed of 100% DOPS. We used tubule diameter and tubule length as two parameters to quantify the strength of the membrane shaping ability of BIN1 NBAR domains. Shown in Fig. 4B, the averaged tubular diameter induced by BIN1 N-BAR WT was 3461 nm, which is comparable to the reported tubule dimensions generated by N-BAR domain proteins. Here, DOPS and PI P2, which are enriched in the inner leaflet of the plasma membrane, provide negative charge promoting electrostatic interactions with the BAR domain while the presence of the smaller headgroup in DOPE lipids results in more lipid packing defects that facilitate insertion of amphipathic helices leading to enhanced tubulation and vesiculation.