The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP_1-19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP_1-19 in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP_1-19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP_1-19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.     

Phospholipid interaction induces molecular-level polymorphism in apolipoprotein C-II amyloid fibrils via alternative assembly pathways

A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.     

Interaction of membrane-bound islet amyloid polypeptide with soluble and crystalline insulin

Islet amyloid polypeptide (IAPP, also known as amylin) is the major protein component of pancreatic amyloid fibers in type II diabetes and is normally cosecreted with insulin from the beta-cells of the pancreas. IAPP forms amyloid fibrils rapidly at concentrations well below those found in vivo, yet progression of type II diabetes occurs over many years. Insulin, a known inhibitor of IAPP fibrillogenesis, exists as a dense crystalline or near-crystalline core in the secretory vesicle, while IAPP localizes to the region between the crystal and the secretory vesicle membrane. In vitro, IAPP fibrillogenesis is both accelerated by lipid membranes and inhibited by monomeric insulin. In this work, we investigate insulin-IAPP-lipid interactions in vitro under conditions chosen to approximate native secretory vesicle physiology and the amyloid disease state. The effect of insulin on IAPP fibrillogenesis is investigated using fluorescence spectrometry. Additionally, interactions of IAPP and lipids with crystalline insulin are studied using fluorescence microscopy. We find that, while soluble states of insulin and IAPP do not interact significantly, large assemblies of either insulin (crystals) or IAPP (fibers) can lead to stable IAPP-insulin interactions. The results raise the possibility of multiple physiological interactions between these two beta-cell hormones.     

Helix Stabilization Precedes Aqueous and Bilayer-Catalyzed Fiber Formation in Islet Amyloid Polypeptide

Islet amyloid polypeptide (IAPP) is an unstructured polypeptide hormone that is cosecreted with insulin. In patients with type 2 diabetes, IAPP undergoes a transition from its natively disordered state to a highly ordered, all-β-strand amyloid fiber. Although predominantly disordered, IAPP transiently samples α-helical structure in solution. IAPP adopts a fully helical structure when bound to membrane surfaces in a process associated with catalysis of amyloid formation. Here, we use spectroscopic techniques to study the structure of full-length, monomeric IAPP under amyloidogenic conditions. We observe that the residues with helical propensity in solution (1-22) also form the membrane-associated helix. Additionally, reduction of the N-terminal disulfide bond (Cys2-Cys7) decreases the extent of helix formed throughout this region. Through manipulation of sample conditions to increase or decrease the amount of helix, we show that the degree of helix formed affects the rate of amyloid assembly. Formation of helical structure is directly correlated with enhanced amyloid formation both on the membrane surface and in solution. These observations support suggested mechanisms in which parallel helix associations bring together regions of the peptide that could nucleate β-strand structure. Remarkably, stabilization of non-amyloid structure appears to be a key intermediate in assembly of IAPP amyloid.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

Morin hydrate inhibits amyloid formation by islet amyloid polypeptide and disaggregates amyloid fibers

The polypeptide hormone Islet Amyloid Polypeptide (IAPP, amylin) is responsible for islet amyloid formation in type-2 diabetes and in islet cell transplants, where it may contribute to graft failure. Human IAPP is extremely amyloidogenic and fewer inhibitors of IAPP amyloid formation have been reported than for the Alzheimer's Aβ peptide or for α-synuclein. The ability of a set of hydroxyflavones to inhibit IAPP amyloid formation was tested. Fluorescence detected thioflavin-T-binding assays are the most popular methods for measuring the kinetics of amyloid formation and for screening potential inhibitors; however, we show that they can lead to false positives with hydroxyflavones. Several of the compounds inhibit thioflavin-T fluorescence, but not amyloid formation; a result which highlights the hazards of relying solely on thioflavin-T assays to screen potential inhibitors. Transmission electron microscopy (TEM) and right-angle light scattering show that Morin hydrate (2',3,4',5,7-Pentahydroxyflavone) inhibits amyloid formation by human IAPP and disaggregates preformed IAPP amyloid fibers. In contrast, Myricetin, Kaempferol, and Quercetin, which differ only in hydroxyl groups on the B-ring, are not effective inhibitors. Morin hydrate represents a new type of IAPP amyloid inhibitor and the results with the other compounds highlight the importance of the substitution pattern on the B-ring.     

Negatively charged phospholipid membranes induce amyloid formation of medin via an alpha-helical intermediate

Medin, a recently discovered 5.5 kDa peptide, is associated with amyloid deposits in the medial layer of human arteries and the prevalence is nearly 100% within individuals above 50 years. Presently, not much is known about its biochemical and biophysical properties or its pathway from soluble peptide to insoluble amyloid. Here we have characterized the behavior of medin in the presence of lipid membranes, using circular dichroism, isothermal titration calorimetry, differential scanning calorimetry, size exclusion chromatography, and atomic force microscopy (AFM). Medin was shown to exist as a monomer in solution with a predominantly random-coil structure. It binds lipid vesicles that have either a neutral or a negative surface potential. Upon association to membranes containing acidic lipids, it undergoes an electrostatically driven conformational change towards a mainly α-helical state. Prolonged incubation converts medin from an α-helical structure into an amyloid β-sheet fibrillar state as confirmed by AFM. Based on these findings, we propose a mechanism of medin-amyloid formation where medin electrostatically associates in its monomeric form to biological interfaces displaying a negative potential. This process both increases the local peptide concentration and induces an aggregation-prone α-helical fold.     

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar β-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, β-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.     

Inhibitors of islet amyloid polypeptide fibrillogenesis, and the treatment of type-2 diabetes

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. Although it may be a secondary event in the etiology of diabetes, the accumulation of insoluble IAPP fibrils is considered to be a primary cause of β-cell failure in affected individuals. A possible means of inhibiting this process is through the use of small peptides that bind to IAPP and prevent fibril polymerization. This approach has been examined using a series of overlapping hexamers that target the known amyloidogenic regions of IAPP. Peptides were examined usingin vitroassays and active inhibitors were identified by their ability to prevent amyloid-related conformational transitions and IAPP aggregation. Fragments such as those corresponding to the IAPP-derived sequences, SNNFGA (residues 20–25) and GAILSS (residues 24–29), were potent inhibitors ofβ-sheet folding and amyloid fibril formation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP significantly decreased the density of fibrils and any remaining structures displayed altered morphology. In some, but not all cases, inhibition of amyloid fibrils also correlated with an ability to reduce IAPP-mediated cytotoxicity as determined in cell culture studies. The results from these studies suggest that these two peptide inhibitors differ in their mechanisms of action possibly due to unique interactions with the full-length IAPP molecule. These inhibitors form the basis of a therapeutic strategy to prevent amyloid accumulation leading to improved islet survival and a potentially novel treatment for type-2 diabetes.     

Promotion of amyloid beta protein misfolding and fibrillogenesis by a lipid oxidation product

Oxidatively damaged lipid membranes are known to promote the aggregation of amyloid β proteins and fibril formation. Oxidative damage typically produces 4-hydroxy-2-nonenal when lipid membranes contain ω-6 polyunsaturated fatty acyl chains, and this compound is known to modify the three His residues in Aβ proteins by Michael addition. In this report, the ability of 4-hydroxy-2-nonenal to reproduce the previously observed amyloidogenic effects of oxidative lipid damage on amyloid β proteins is demonstrated and the mechanism by which it exerts these effects is examined. Results indicate that 4-hydroxy-2-nonenal modifies the three His residues in amyloid beta proteins, which increases their membrane affinity and causes them to adopt a conformation on membranes that is similar to their conformation in a mature amyloid fibril. As a consequence, fibril formation is accelerated at relatively low protein concentrations, and the ability to seed the formation of fibrils by unmodified amyloid beta proteins is enhanced. These in vitro findings linking oxidative stress to amyloid fibril formation may be significant to the in vivo mechanism by which oxidative stress is linked to the formation of amyloid plaques in Alzheimer's disease.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

Preferential accumulation of Aβ(1−42) on gel phase domains of lipid bilayers: An AFM and fluorescence study

Peptide-membrane interactions have been implicated in both the toxicity and aggregation of β-amyloid (Aβ) peptides. Recent studies have provided evidence for the involvement of liquid-ordered membrane domains known as lipid rafts in the formation and aggregation of Aβ. As a model, we have examined the interaction of Aβ(1−42) with phase separated DOPC/DPPC lipid bilayers using a combination of atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRF). AFM images show that addition of Aβ to preformed supported bilayers leads to accumulation of small peptide aggregates exclusively on the gel phase DPPC domains. Initial aggregates are observed approximately 90 min after peptide addition and increase in diameter to 45-150 nm within 24 h. TIRF studies with a mixture of Aβ and Aβ-Fl demonstrate that accumulation of the peptide on the gel phase domains occurs as early as 15 min after Aβ addition and is maintained for over 24 h. By contrast, Aβ is randomly distributed throughout both fluid and gel phases when the peptide is reconstituted into DOPC/DPPC vesicles prior to formation of a supported bilayer. The preferential accumulation of Aβ on DPPC domains suggests that rigid domains may act as platforms to concentrate peptide and enhance its aggregation and may be relevant to the postulated involvement of lipid rafts in modulating Aβ activity in vivo.     

Antimicrobial peptides temporins B and L induce formation of tubular lipid protrusions from supported phospholipid bilayers

The binding of the antimicrobial peptides temporins B and L to supported lipid bilayer (SLB) model membranes composed of phosphatidylcholine and phosphatidylglycerol (4:1, mol/mol) caused the formation of fibrillar protrusions, visible by fluorescent microscopy of both a fluorescent lipid analog and a labeled peptide. Multicolor imaging at low peptide-to-lipid ratios (P/L < approximately 1:5) revealed an initial in-plane segregation of membrane-bound peptide and partial exclusion of lipid from the peptide-enriched areas. Subsequently, at higher P/L numerous flexible lipid fibrils were seen growing from the areas enriched in lipid. The fibrils have diameters <250 nm and lengths of up to approximately 1 mm. Fibril formation reduces the in-plane heterogeneity and results in a relatively even redistribution of bound peptide over the planar bilayer and the fibrils. Physical properties of the lipid fibrils suggest that they have a tubular structure. Our data demonstrate that the peptide-lipid interactions alone can provide a driving force for the spontaneous membrane shape transformations leading to tubule outgrowth and elongation. Further experiments revealed the importance of positive curvature strain in the tubulation process as well as the sufficient positive charge on the peptide (>/=+2). The observed membrane transformations could provide a simplified in vitro model for morphogenesis of intracellular tubular structures and intercellular connections.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

Structural rearrangement of model membranes by the peptide antibiotic NK-2

We have developed a novel α-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 °C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.     

The two-fold aspect of the interplay of amyloidogenic proteins with lipid membranes

Investigating the pathways leading to the formation of amyloid protein aggregates and the mechanism of their cytotoxicity is fundamental for a deeper understanding of a broad range of human diseases. Increasing evidence indicates that early aggregates are responsible for the cytotoxic effects. This paper addresses the catalytic role of lipid surfaces in promoting aggregation of amyloid proteins and the permeability changes that these aggregates induce on lipid membranes. Effects of amyloid aggregates on model systems such as monolayers, vesicles, liposomes and supported lipid bilayers are reviewed. In particular, the relevance of atomic force microscopy in detecting both kinetics of amyloid formation and amyloid-membrane interactions is emphasized.     

Optimized experimental pre-treatment strategy for temporary inhibition of islet amyloid polypeptide aggregation

Islet amyloid polypeptide (IAPP) is a neuroendocrine hormone from pancreatic β-cells. Misfolded, aggregated IAPP is believed to be toxic to islet cells and amyloid deposits in the pancreas are pathological hallmarks of type 2 diabetes. Rapid fibrillization of this peptide makes it difficult to study in its soluble form, impeding a better understanding of its role. In this study, a variety of popular pretreatment methods were tested for their ability to delay aggregation of IAPP, including solutions of hexafluoroisopropanol, sodium hydroxide, hydrochloric acid, phosphate buffered saline, ammonium hydroxide, as well as tris buffer at different pH and containing either calcium (II), zinc (II), or iron (II). Aggregation was assessed using the thioflavin T fluorescence assay as well as by transmission electron microscopy. Tris buffer at pH 8.1 containing Zn(II) was found to have the best balance of temporary inhibition of aggregation and biological relevance.     

Detergent-like actions of linear amphipathic cationic antimicrobial peptides

Antimicrobial peptides have raised much interest as pathogens become resistant against conventional antibiotics. We review biophysical studies that have been performed to better understand the interactions of linear amphipathic cationic peptides such as magainins, cecropins, dermaseptin, δ-lysin or melittin. The amphipathic character of these peptides and their interactions with membranes resemble the properties of detergent molecules and analogies between membrane-active peptide and detergents are presented. Several models have been suggested to explain the pore-forming, membrane-lytic and antibiotic activities of these peptides. Here we suggest that these might be ‘special cases’ within complicated phase diagrams describing the morphological plasticity of peptide/lipid supramolecular assemblies.     

Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. ³¹P and ²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.