ABSTRACTThe N‐terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33–55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild‐type AdVpVI (33–55) (WT) and phenylalanine‐substituted peptides (L40F) demonstrated that both peptides have positive membrane‐curvature‐inducing ability. These peptides showed higher binding affinity to 50‐nm large unilamellar vesicles (LUVs) than to 200‐nm LUVs. However, no enhanced perturbation by these peptides was observed for 50‐nm LUVs compared to 200‐nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50‐nm LUVs than for 200‐nm LUVs, which can be attributed to possible shallower binding of L40F on membranes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430–439, 2016.