The role of hydrophobic patches of de novo designed MSI-78 and VG16KRKP antimicrobial peptides on fragmenting model bilayer membranes.

Antimicrobial peptides (AMPs) with cell membrane lysing capability are considered potential candidates for the development of the next generation of antibiotics. Designing novel AMPs requires an in-depth understanding of the mechanism of action of the peptides. In this work, we used various biophysical techniques including 31P solid-state NMR to examine the interaction of model membranes with amphipathic de novo-designed peptides. Two such peptides, MSI-78 and VG16KRKP, were designed with different hydrophobicity and positive charges. The model lipid membranes were constituted by mixing lipids of varying degrees of 'area per lipid' (APL), which directly affected the packing properties of the membrane. The observed emergence of the isotropic peak in 31P NMR spectra as a function of time is a consequence of the fragmentation of the membrane mediated by the peptide interaction. The factors such as the charges, overall hydrophilicity of the AMPs, as well as lipid membrane packing, contributed to the kinetics of membrane fragmentation. Furthermore, we anticipate the designed AMPs follow the carpet and toroidal pore mechanisms when lysing the cell membrane. This study highlights the significance of the effect of the overall charges and the hydrophobicity of the novel AMPs designed for antimicrobial activity.

Structural insights into the interaction of antifungal peptides and ergosterol containing fungal membrane.

The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation.

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis.

Comparison of Synthetic Neuronal Model Membrane Mimics in Amyloid Aggregation at Atomic Resolution.

Alzheimer's disease (AD) is a severe neurodegenerative disorder caused by abnormal accumulation of toxic amyloid plaques of the amyloid-beta (Aß) or the tau proteins in the brain. The plaque deposition leading to the collapse of the cellular integrity is responsible for a myriad of surface phenomena acting at the neuronal lipid interface. Recent years have witnessed dysfunction of the blood-brain barriers (BBB) associated with AD. Several studies support the idea that BBB acts as a platform for the formation of misfolded Aß peptide, promoting oligomerization and fibrillation, compromising the overall integrity of the central nervous system. While the amyloid plaque deposition has been known to be responsible for the collapse of the BBB membrane integrity, the causal effect relationship between BBB and Aß amyloidogenesis remains unclear. In this study, we have used physiologically relevant synthetic model membrane systems to gain atomic insight into the functional aspects of the lipid interface. Here, we have used a minimalist BBB mimic, POPC/POPG/cholesterol/GM1, to compare with the native BBB (total lipid brain extract (TLBE)), to understand the molecular events occurring in the membrane-induced Aß40 amyloid aggregation. Our study showed that the two membrane models accelerated the Aß40 aggregation kinetics with differential secondary structural transitions of the peptide. The observed structural transitions are defined by the lipid compositions, which in turn undermines the differences in lipid surface phenomena, leading to peptide induced cellular toxicity in the neuronal membrane.

Magnetic Alignment of Polymer Nanodiscs Probed by Solid-State NMR Spectroscopy.

The ability of amphipathic polymers to self-assemble with lipids and form nanodiscs has been a boon for the field of functional reconstitution of membrane proteins. In a field dominated by detergent micelles, a unique feature of polymer nanodiscs is their much-desired ability to align in the presence of an external magnetic field. Magnetic alignment facilitates the application of solid-state nuclear magnetic resonance (NMR) spectroscopy and aids in the measurement of residual dipolar couplings via well-established solution NMR spectroscopy. In this study, we comprehensively investigate the magnetic alignment properties of styrene maleimide quaternary ammonium (SMA-QA) polymer-based nanodiscs by using 31P and 14N solid-state NMR experiments under static conditions. The results reported herein demonstrate the spontaneous magnetic alignment of large-sized (≥20 nm diameter) SMA-QA nanodiscs (also called as macro-nanodiscs) with the lipid bilayer normal perpendicular to the magnetic field direction. Consequently, the orientation of macro-nanodiscs is further shown to flip the alignment axis parallel to the magnetic field direction upon the addition of a paramagnetic lanthanide salt. These results demonstrate the use of SMA-QA polymer nanodiscs for solid-state NMR applications including structural studies on membrane proteins.

Structural insights into the combinatorial effects of antimicrobial peptides reveal a role of aromatic-aromatic interactions in antibacterial synergism.

The recent development of plants that overexpress antimicrobial peptides (AMPs) provides opportunities for controlling plant diseases. Because plants employ a broad-spectrum antimicrobial defense, including those based on AMPs, transgenic modification for AMP overexpression represents a potential way to utilize a defense system already present in plants. Herein, using an array of techniques and approaches, we report on VG16KRKP and KYE28, two antimicrobial peptides, which in combination exhibit synergistic antimicrobial effects against plant pathogens and are resistant against plant proteases. Investigating the structural origin of these synergistic antimicrobial effects with NMR spectroscopy of the complex formed between these two peptides and their mutated analogs, we demonstrate the formation of an unusual peptide complex, characterized by the formation of a bulky hydrophobic hub, stabilized by aromatic zippers. Using three-dimensional structure analyses of the complex in bacterial outer and inner membrane components and when bound to lipopolysaccharide (LPS) or bacterial membrane mimics, we found that this structure is key for elevating antimicrobial potency of the peptide combination. We conclude that the synergistic antimicrobial effects of VG16KRKP and KYE28 arise from the formation of a well-defined amphiphilic dimer in the presence of LPS and also in the cytoplasmic bacterial membrane environment. Together, these findings highlight a new application of solution NMR spectroscopy to solve complex structures to study peptide-peptide interactions, and they underscore the importance of structural insights for elucidating the antimicrobial effects of AMP mixtures.

Role of non-electrostatic forces in antimicrobial potency of a dengue-virus derived fusion peptide VG16KRKP: Mechanistic insight into the interfacial peptide-lipid interactions.

Cationic antimicrobial peptides (AMPs) are emerging as effective alternatives to conventional therapeutics that are used against the ever-rising number of multidrug-resistant microbial strains. Most studies established the peptide's amphipathicity and electrostatic interaction with the membrane as the basis for their antimicrobial effect. However, the interplay between the stoichiometric ratio of lipids, local geometry, diverse physicochemical properties of the host membranes and antimicrobial peptide efficacy is still poorly understood. In the present study, we investigate the mechanism of interaction of VG16KRKP (VARGWKRKCPLFGKGG), a novel AMP designed from the dengue-virus fusion peptide, with bacterial/fungal membrane mimics. Fluorescence based dye leakage assays show that membrane disruption is not solely induced by electrostatic interaction but also driven by stoichiometric ratio of the lipids that dictates the net surface charge, amount of lipid defects and local geometry of the membrane. Solid-state 14N and 31P NMR experiments show that peptide interaction results in lowering of lipid order around both the headgroups and acyl chains, suggesting deep peptide insertion. Further, an increase or decrease in membrane stability of the host membrane was observed in differential scanning calorimetry (DSC) thermograms, dictated by the overall stoichiometric ratio of the lipids and the sterol present. In general, our results help understand the diverse fates of host membranes against an antimicrobial peptide.

A blend of two resveratrol derivatives abolishes hIAPP amyloid growth and membrane damage.

Type II diabetes mellitus (T2DM) is characterized by the presence of amyloid deposits of the human islet amyloid polypeptide (hIAPP) in pancreatic ß-cells. A wealth of data supports the hypothesis that hIAPP's toxicity is related to an abnormal interaction of amyloids with islet cell membranes. Thus, many studies aimed at finding effective therapies for T2DM focus on the design of molecules that are able to inhibit hIAPP's amyloid growth and the related membrane damage as well. Based on this view and inspired by its known anti-amyloid properties, we have functionalized resveratrol with a phosphoryl moiety (4'-O-PR) that improves its solubility and pharmacological properties. A second resveratrol derivative has also been obtained by conjugating resveratrol with a dimyristoylphosphatidyl moiety (4'-DMPR). The use of both compounds resulted in abolishing both amyloid growth and amyloid mediated POPC/POPS membrane damage in tube tests. We propose that a mixture of a water-soluble anti-aggregating compound and its lipid-anchored derivative may be employed as a general strategy to prevent and/or to halt amyloid-related membrane damage.

Insulin-eukaryotic model membrane interaction: Mechanistic insight of insulin fibrillation and membrane disruption.

Injection of exogenous insulin in the subcutaneous mass has been a proven therapy for type II diabetes. However, chronic administration of insulin often develops local amyloidosis at the injection site, pathologically known as "Insulin Ball". This reduces the insulin bioavailability and exacerbates the disease pathology. Thus, the molecular interaction between insulin and the recipient's membrane surface plays a co-operative role in accelerating the amyloidosis. This interaction, however, is different from the molecular interaction of insulin with the native membranous environment of the pancreatic ß-cells. The differential membrane mediated interaction that directly affects the aggregation kinetics of insulin remains elusive yet intriguing to understand the mechanism of pathological development. In this study we have characterized the interactions of insulin at different states with model eukaryotic membranes using high and low-resolution spectroscopic techniques in combination with microscopic investigation. Our results show that insulin amyloid intermediates are capable of interacting with model membranes with variable functional affinity towards the different compositions. Fluorescence correlation spectroscopy confirms the aggregation states of insulin in presence of the eukaryotic model membranes while solid-state NMR spectroscopy in conjugation with differential scanning calorimetry elucidates the molecular interaction of insulin intermediates with the lipid head groups along with the acyl chains. Additionally, dye leakage assays support the eukaryotic model membrane disruption by insulin intermediates, similar to hIAPP and Aß40, as previously reported. Thus, the present study establishes the distinct mode of interactions of insulin amyloid with pancreatic ß-cell and general mammalian cell mimicking membranes.

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans.

There is a significant need for developing compounds that kill Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis in immunocompromised individuals. Here, we report the mode of action of a designed antifungal peptide, VG16KRKP (VARGWKRKCPLFGKGG) against C. neoformans. It is shown that VG16KRKP kills fungal cells mainly through membrane compromise leading to efflux of ions and cell metabolites. Intracellular localization, inhibition of in vitro transcription, and DNA binding suggest a secondary mode of action for the peptide, hinting at possible intracellular targets. Atomistic structure of the peptide determined by NMR experiments on live C. neoformans cells reveals an amphipathic arrangement stabilized by hydrophobic interactions among A2, W5, and F12, a conventional folding pattern also known to play a major role in peptide-mediated Gram-negative bacterial killing, revealing the importance of this motif. These structural details in the context of live cell provide valuable insights into the design of potent peptides for effective treatment of human and plant fungal infections.

Detergent-type membrane fragmentation by MSI-78, MSI-367, MSI-594, and MSI-843 antimicrobial peptides and inhibition by cholesterol: a solid-state nuclear magnetic resonance study.

Multidrug resistance against the existing antibiotics is becoming a global threat, and any potential drug that can be designed using cationic antimicrobial peptides (AMP) could be an alternate solution to alleviate this existing problem. The mechanism of action of killing bacteria by an AMP differs drastically in comparison to that of small molecule antibiotics. The main target of AMPs is to interact with the lipid bilayer of the cell membrane and disrupt it to kill bacteria. Consequently, the modes of membrane interaction that lead to the selectivity of an AMP are very important to understand. Here, we have used different membrane compositions, such as negatively charged, zwitterionic, or mixed large unilamellar vesicles (LUVs), to study the interaction of four different synthetically designed cationic, linear antimicrobial peptides: MSI-78 (commercially known as pexiganan), MSI-367, MSI-594, and MSI-843. Our solid-state nuclear magnetic resonance (NMR) experiments confirmed that the MSI peptides fragmented LUVs through a detergent-like carpet mechanism depending on the amino acid sequence of the MSI peptide and/or the membrane composition of LUVs. Interestingly, the fragmented lipid aggregates such as SUVs or micelles are sufficiently small to produce an isotropic peak in the (31)P NMR spectrum. These fragmented lipid aggregates contain only MSI peptides bestowed with lipid molecules as confirmed by NMR in conjunction with circular dichroism spectroscopy. Our results also demonstrate that cholesterol, which is present only in the eukaryotic cell membrane, inhibits the MSI-induced fragmentation of LUVs, suggesting that the MSI peptides can discriminate the bacteria and the eukaryotic cell membranes, and this selectivity could be used for further development of novel antibiotics.

Temperature-resistant bicelles for structural studies by solid-state NMR spectroscopy.

Three-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between -15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from (31)P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.

Synthetic Flavonoids, Aminoisoflavones: Interaction and Reactivity with Metal-Free and Metal-Associated Amyloid-ß Species.

Metal ion homeostasis in conjunction with amyloid-ß (Aß) aggregation in the brain has been implicated in Alzheimer's disease (AD) pathogenesis. To uncover the interplay between metal ions and Aß peptides, synthetic, multifunctional small molecules have been employed to modulate Aß aggregation in vitro. Naturally occurring flavonoids have emerged as a valuable class of compounds for this purpose due to their ability to modulate both metal-free and metal-induced Aß aggregation. Although, flavonoids have shown anti-amyloidogenic effects, the structural moieties of flavonoids responsible for such reactivity have not been fully identified. In order to understand the structure-interaction-reactivity relationship within the flavonoid family for metal-free and metal-associated Aß, we designed, synthesized, and characterized a set of isoflavone derivatives, aminoisoflavones (1-4), that displayed reactivity (i.e., modulation of Aß aggregation) in vitro. NMR studies revealed a potential binding site for aminoisoflavones between the N-terminal loop and central helix on prefibrillar Aß different from the non-specific binding observed for other flavonoids. The absence or presence of the catechol group differentiated the binding affinities and enthalpy/entropy balance between aminoisoflavones and Aß. Furthermore, having a catechol group influenced the binding mode with fibrillar Aß. Inclusion of additional substituents moderately tuned the impact of aminoisoflavones on Aß aggregation. Overall, through these studies, we obtained valuable insights on the requirements for parity among metal chelation, intermolecular interactions, and substituent variation for Aß interaction.

A simple colorimetric chemosensor bearing a carboxylic acid group with high selectivity for CN-.

A new simple 'naked eye' chemosensor 1 (sodium (E)-2-((2-(3-hydroxy-2-naphthoyl)hydrazono)methyl)benzoate) has been synthesized for detection of CN- in a mixture of DMF/H2O (9:1). The sensor 1 comprises of a naphthoic hydrazide as efficient hydrogen bonding donor group and a benzoic acid as the moiety with the water solubility. The receptor 1 showed high selectivity toward cyanide ions in a 1:1 stoichiometric manner, which induces a fast color change from colorless to yellow for CN- over other anions. Therefore, receptor 1 could be useful for cyanide detection in aqueous environment, displaying a high distinguishable selectivity from hydrogen bonded anions and being clearly visible to the naked eye.

Tuning reactivity of diphenylpropynone derivatives with metal-associated amyloid-ß species via structural modifications.

A diphenylpropynone derivative, DPP2, has been recently demonstrated to target metal-associated amyloid-ß (metal-Aß) species implicated in Alzheimer's disease (AD). DPP2 was shown to interact with metal-Aß species and subsequently control Aß aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aß species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (C1/C2, P1/P2, and PA1/PA2) as structurally modified DPP2 analogues. A similar metal binding site to that of DPP2 was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aß species, and metal-Aß species. Distinct reactivities of our chemical family toward in vitro Aß aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (C2) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aß aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with DPP2. Lastly, modifications on the DPP framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of DPP2 could govern different metal binding properties, interactions with Aß and metal-Aß species, reactivity toward metal-free and metal-induced Aß aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal-Aß species and modulating their reactivity in biological systems.

Lipid composition-dependent membrane fragmentation and pore-forming mechanisms of membrane disruption by pexiganan (MSI-78).

The potency and selectivity of many antimicrobial peptides (AMPs) are correlated with their ability to interact with and disrupt the bacterial cell membrane. In vitro experiments using model membranes have been used to determine the mechanism of membrane disruption of AMPs. Because the mechanism of action of an AMP depends on the ability of the model membrane to accurately mimic the cell membrane, it is important to understand the effect of membrane composition. Anionic lipids that are present in the outer membrane of prokaryotes but are less common in eukaryotic membranes are usually thought to be key for the bacterial selectivity of AMPs. We show by fluorescence measurements of peptide-induced membrane permeabilization that the presence of anionic lipids at high concentrations can actually inhibit membrane disruption by the AMP MSI-78 (pexiganan), a representative of a large class of highly cationic AMPs. Paramagnetic quenching studies suggest MSI-78 is in a surface-associated inactive mode in anionic sodium dodecyl sulfate micelles but is in a deeply buried and presumably more active mode in zwitterionic dodecylphosphocholine micelles. Furthermore, a switch in mechanism occurs with lipid composition. Membrane fragmentation with MSI-78 can be observed in mixed vesicles containing both anionic and zwitterionic lipids but not in vesicles composed of a single lipid of either type. These findings suggest membrane affinity and membrane permeabilization are not always correlated, and additional effects that may be more reflective of the actual cellular environment can be seen as the complexity of the model membranes is increased.

Two-step mechanism of membrane disruption by Aß through membrane fragmentation and pore formation.

Disruption of cell membranes by Aß is believed to be one of the key components of Aß toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aß occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aß(1-40), defects form on the membrane that share many of the properties of Aß channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aß(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aß and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.

Phosphatidylethanolamine enhances amyloid fiber-dependent membrane fragmentation.

The toxicity of amyloid-forming peptides has been hypothesized to reside in the ability of protein oligomers to interact with and disrupt the cell membrane. Much of the evidence for this hypothesis comes from in vitro experiments using model membranes. However, the accuracy of this approach depends on the ability of the model membrane to accurately mimic the cell membrane. The effect of membrane composition has been overlooked in many studies of amyloid toxicity in model systems. By combining measurements of membrane binding, membrane permeabilization, and fiber formation, we show that lipids with the phosphatidylethanolamine (PE) headgroup strongly modulate the membrane disruption induced by IAPP (islet amyloid polypeptide protein), an amyloidogenic protein involved in type II diabetes. Our results suggest that PE lipids hamper the interaction of prefibrillar IAPP with membranes but enhance the membrane disruption correlated with the growth of fibers on the membrane surface via a detergent-like mechanism. These findings provide insights into the mechanism of membrane disruption induced by IAPP, suggesting a possible role of PE and other amyloids involved in other pathologies.

Role of cationic group structure in membrane binding and disruption by amphiphilic copolymers.

Cationic, amphiphilic polymers are currently being used as antimicrobial agents that disrupt biomembranes, although their mechanisms remain poorly understood. Herein, membrane association and disruption by amphiphilic polymers bearing primary, tertiary, or quaternary ammonium salt groups reveal the role of cationic group structure in the polymer-membrane interaction. The dissociation constants of polymers to liposomes of POPC were obtained by a fluorometric assay, exploiting the environmental sensitivity of dansyl moieties in the polymer end groups. Dye leakage from liposomes and solid-state NMR provided further insights into the polymer-induced membrane disruption. Interestingly, the polymers with primary amine groups induced reorganization of the bilayer structure to align lipid headgroups perpendicular to the membrane. The results showed that polymers bearing primary amines exceed the tertiary and quaternary ammonium counterparts in membrane binding and disrupting abilities. This is likely due to enhanced complexation of primary amines to the phosphate groups in the lipids, through a combination of hydrogen bonding and electrostatic interactions.

Limiting an antimicrobial peptide to the lipid-water interface enhances its bacterial membrane selectivity: a case study of MSI-367.

In a minimalist design approach, a synthetic peptide MSI-367 [(KFAKKFA)(3)-NH(2)] was designed and synthesized with the objective of generating cell-selective nonlytic peptides, which have a significant bearing on cell targeting. The peptide exhibited potent activity against both bacteria and fungi, but no toxicity to human cells at micromolar concentrations. Bacterial versus human cell membrane selectivity of the peptide was determined via membrane permeabilization assays. Circular dichroism investigations revealed the intrinsic helix propensity of the peptide, ß-turn structure in aqueous buffer and extended and turn conformations upon binding to lipid vesicles. Differential scanning calorimetry experiments with 1,2-dipalmitoleoyl-sn-glycero-3-phosphatidylethanolamine bilayers indicated the induction of positive curvature strain and repression of the fluid lamellar to inverted hexagonal phase transition by MSI-367. Results of isothermal titration calorimetry (ITC) experiments suggested the possibility of formation of specific lipid-peptide complexes leading to aggregation. (2)H nuclear magnetic resonance (NMR) of deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) multilamellar vesicles confirmed the limited effect of the membrane-embedded peptide at the lipid-water interface. (31)P NMR data indicated changes in the lipid headgroup orientation of POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine lipid bilayers upon peptide binding. Membrane-embedded and membrane-inserted states of the peptide were observed via sum frequency generation vibrational spectroscopy. Circular dichroism, ITC, and (31)P NMR data for Escherichia coli lipids agree with the hypothesis that strong electrostatic lipid-peptide interactions embrace the peptide at the lipid-water interface and provide the basis for bacterial cell selectivity.