In this work, we have identified an additional functional motif of MinE that is associated with MinE-induced membrane deformation. We have provided direct evidence that the extreme Nterminus of MinE from E. coli folds into an amphipathic a-helix when associated with a membrane. This property differed from MinE from Neisseria gonorrhoeae , which showed a stable Nterminal helix in solution . Meanwhile, we have further monitored MinE-induced membrane deformation using in vitro systems of synthetic giant liposomes and supported lipid bilayers via time-lapse fluorescence microscopy. This MinE-induced membrane deformation required both the earlier identified charged residues R10, K11, and K12 and the amphipathic motif identified in this report. Disturbing the amphipathicity in this region not only led to failure to deform the membrane in vitro, but also caused alterations in protein stability, which may serve as a control mechanism for the regulation of the cellular concentration of MinE. In summary, this study of MinE illustrates the universal mechanisms involved in the targeting of peripheral membrane proteins that are capable of causing membrane deformation; such mechanisms have prokaryotic and eukaryotic origins. To investigate whether other mechanisms besides the electrostatic interaction are involved in mediating the MinE-induced membrane deformation, we analyzed the MinE protein sequence using helical wheel projection programs. We found that residues 2�C9 were capable of forming an amphipathic helix of 1�C2 helical turns . Residues A2, L3, L4, F6, F7, and L8 formed a large non-polar, hydrophobic face, and residues D5 and S9 were located on a hydrophilic surface. The extreme N-terminus of MinE from 11 other bacterial species showed propensities to form amphipathic helices, and had 4�C6 residues located on a hydrophobic surface . The high conservation of amphipathic helix formation was suggestive of its importance, and led us to hypothesize that this amphipathic helix, along with the basic residues R10, K11, and K12 , served as a membrane anchor that sustains the peripheral association of MinE. To explore this hypothesis, we took advantage of the characteristic spectral shift of tryptophan fluorescence emission that occurs as a function of solvent polarity and serves as a measure of peptide-membrane interactions . A single tryptophan substitution was introduced in MinE1�C31 during 129-56-6 peptide synthesis to replace residues A2, L3, L4, F6, F7, or L8. A tryptophan Epoxomicin residue added to the C-terminus of MinE1-31 served as a control. To further investigate the helix forming ability of MinE and its association with the membrane, we measured the far-UV circular dichroism spectra of MinE1-12 and MinE1-31 in the presence or absence of liposomes .