Mechanism Of Membrane Disruption Research Articles

Mechanisms Of Antimicrobial Peptide Action Determined Using Chemical And Collisional Quenching Assays

Peptide-lipid interactions are pertinent to antimicrobial peptides (AMP) activity, stringency, and selectivity. The ability of AMPs to disrupt the target cell's lipid bilayer has been described using the nonspecific “carpet” model or using models that assume stable transbilayer pores (e.g. barrel stave pores). With the carpet model, there is a strong electrostatic and hydrophobic attraction between peptide and the lipid interfacial groups such that the peptides blanket the cell membrane. Carpet model peptides can then kill the organism by disrupting the lipid bilayer and causing loss of cellular contents. The stable pore model consists of fixed transmembrane structures that rely on an amphipathic amino acid sequence to form membrane-spanning pores. Included with these models, peptides can also induce flip-flop of bilayer lipids or form transient pores. We have developed chemical and collisional quenching assays that help determine the mode of lipid disruption associated with naturally occurring and synthetically designed AMPs. The assays require large unilamellar vesicles (LUVs) with fluorophore-attached lipid head groups in both inner and outer leaflets of the bilayer. Only peptides with stable pore-forming or detergent-like activity allow quenchers access to the inner leaflet. The combination of these quenching assays with leakage experiments and cryo-electron microscopy allows for a more complete description of the mechanism of membrane disruption by peptides.

High-Resolution Structure of Piscidin in Aligned Lipid Bilayers: Implications for Antimicrobial Mode of Action

Piscidin is an amphipathic cationic antimicrobial peptide that belongs to a large family of versatile host-defense peptides, which interact, at least initially, with cell membranes in order to perform their function. In the research presented here, we characterize the secondary structure and dynamics of piscidin in order to identify factors optimizing specific molecular interactions that are directly related to its function and mode of action. Our long term goal is to identify common principles that will facilitate the design of pharmaceuticals with broad-spectrum antibacterial activity, minimum induction of bacterial resistance, and low toxicity for mammalian cells.Previously, we demonstrated that membrane-bound piscidin 1 (p1) adopts an alpha-helical structure, which lies in the plane of the bilayer and experiences fast motions. Here, high-resolution solid-state NMR spectra have been obtained from multiply 15N-backbone-labeled p1 aligned in hydrated lipid bilayers. Analysis of data from twenty sites of this 22-mer reveals two helical segments separated by a kink at Gly13. This kink may help one portion of the peptide insert more deeply in the hydrophobic lipid bilayer, which may be related to the mechanism of membrane disruption. To characterize water exposure, hydrogen-deuterium exchange experiments have been performed on 15N-backbone labeled samples. In addition, solid-state NMR was applied to 15N-side-chain-labeled His-17 p1 to titrate this side chain, which resides at the interface between the hydrophilic and hydrophobic domains of p1. Overall, our atomic-level investigation of the structure, dynamics, and water exposure of membrane-bound piscidin provides new insights into its mode of action and thus help understand how antimicrobial peptides recognize membranes and initiate their activities on microbial cells.

Effect of Antimicrobial Peptides from Australian Tree Frogs on Anionic Phospholipid Membranes

Skin secretions of Australian tree frogs contain antimicrobial peptides that form part of the host defence mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al., (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1 and caerin 1.1, which exist as random coil in aqueous solution, but have α-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC (d54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid head groups by deuterium and 31P NMR, respectively. We demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterised using surface plasmon resonance (SPR) spectroscopy and solid-state NMR spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides have significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.

Isocryptomerin, a novel membrane-active antifungal compound from Selaginella tamariscina

Isocryptomerin is a biflavonoid isolated from Selaginella tamariscina used in traditional medicine. In this study, we investigated novel antifungal properties of isocryptomerin. The results indicated that isocryptomerin exerted antifungal activity in an energy-independent manner without remarkable hemolytic effects. To understand mode of action(s) of isocryptomerin, we conducted experiments on Candida albicans, a noted human pathogenic fungal strain. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC 4(3)], a translational membrane potential dye, regeneration test with fungal protoplasts, and fluorescence analysis with 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe for membrane studies by depolarization, indicated that isocryptomerin could depolarize fungal plasma membrane. In conclusion, the results suggested that the antifungal activities of isocryptomerin might be due to its membrane-disruption mechanism(s).

The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm

Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.

Purification and characterization of a thermal stable antimicrobial protein from housefly larvae,Musca domestica, induced by ultrasonic wave

This work describes the induction, purification and partial biochemical characterizations of an antimicrobial protein from the housefly larvae induced by ultrasonic wave. It has been purified to apparent homogeneity by ammonium sulfate precipitation followed by Sephadex G-75, Bio-gel P6 gel filtration, and CM-Sepharose Fast Flow cation exchange chromatography. The protein is a cationic protein with an apparent molecular weight of 16315 Da determined by no-denaturing electrophoresis and SDS-PAGE, respectively. Biochemical profile assays show that this protein has good thermal stability, and repeatedly frozen and defrosted durability. The optimum pH for antimicrobial activity is around pH5. The antimicrobial range of the protein includes Gram-positive, Gram-negative bacteria and some fungi. Results of the membrane permeability assays suggest that the probable mode of action of this protein is membrane-disrupting mechanism.

Effect of Antimicrobial Peptides from Australian Tree Frogs on Anionic Phospholipid Membranes

Skin secretions of numerous Australian tree frogs contain antimicrobial peptides that form part of the host defense mechanism against bacterial infection. The mode of action of these antibiotics is thought to be lysis of infectious organisms via cell membrane disruption, on the basis of vesicle-encapsulated dye leakage data [Ambroggio et al. (2005) Biophys. J. 89, 1874-1881]. A detailed understanding of the interaction of these peptides with bacterial membranes at a molecular level, however, is critical to their development as novel antibacterial therapeutics. We focus on four of these peptides, aurein 1.2, citropin 1.1, maculatin 1.1, and caerin 1.1, which exist as random coil in aqueous solution but have alpha-helical secondary structure in membrane mimetic environments. In our earlier solid-state NMR studies, only neutral bilayers of the zwitterionic phospholipid dimyristoylphosphatidylcholine (DMPC) were used. Deuterated DMPC ( d 54-DMPC) was used to probe the effect of the peptides on the order of the lipid acyl chains and dynamics of the phospholipid headgroups by deuterium and (31)P NMR, respectively. In this report we demonstrate several important differences when anionic phospholipid is included in model membranes. Peptide-membrane interactions were characterized using surface plasmon resonance (SPR) spectroscopy and solid-state nuclear magnetic resonance (NMR) spectroscopy. Changes in phospholipid motions and membrane binding information provided additional insight into the action of these antimicrobial peptides. While this set of peptides has significant C- and N-terminal sequence homology, they vary in their mode of membrane interaction. The longer peptides caerin and maculatin exhibited properties that were consistent with transmembrane insertion while citropin and aurein demonstrated membrane disruptive mechanisms. Moreover, aurein was unique with greater perturbation of neutral versus anionic membranes. The results are consistent with a surface interaction for aurein 1.2 and pore formation rather than membrane lysis by the longer peptides.

The MACPF/CDC family of pore-forming toxins

Pore-forming toxins (PFTs) are commonly associated with bacterial pathogenesis. In eukaryotes, however, PFTs operate in the immune system or are deployed for attacking prey (e.g. venoms). This review focuses upon two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF). CDCs are produced by Gram-positive bacteria and lyse or permeabilize host cells or intracellular organelles during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles and function in immunity, invasion and development. The structure and molecular mechanism of several CDCs are relatively well characterized. Pore formation involves oligomerization and assembly of soluble monomers into a ring-shaped pre-pore which undergoes conformational change to insert into membranes, forming a large amphipathic transmembrane β-barrel. In contrast, the structure and mechanism of MACPF proteins has remained obscure. Recent crystallographic studies now reveal that although MACPF and CDCs are extremely divergent at the sequence level, they share a common fold. Together with biochemical studies, these structural data suggest that lytic MACPF proteins use a CDC-like mechanism of membrane disruption, and will help understand the roles these proteins play in immunity and development.

Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane

Fibrillar protein deposits (amyloid) in the pancreatic islets of Langerhans are thought to be involved in death of the insulin-producing islet beta cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this beta cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins.

Dual Mechanism of Bacterial Lethality for a Cationic Sequence-Random Copolymer that Mimics Host-Defense Antimicrobial Peptides

Flexible sequence-random polymers containing cationic and lipophilic subunits that act as functional mimics of host-defense peptides have recently been reported. We used bacteria and lipid vesicles to study one such polymer, having an average length of 21 residues, that is active against both Gram-positive and Gram-negative bacteria. At low concentrations, this polymer is able to permeabilize model anionic membranes that mimic the lipid composition of Escherichia coli, Staphylococcus aureus, or Bacillus subtilis but is ineffective against model zwitterionic membranes, which explains its low hemolytic activity. The polymer is capable of binding to negatively charged vesicles, inducing segregation of anionic lipids. The appearance of anionic lipid-rich domains results in formation of phase-boundary defects through which leakage can occur. We had earlier proposed such a mechanism of membrane disruption for another antimicrobial agent. Experiments with the mutant E. coli ML-35p indicate that permeabilization is biphasic: at low concentrations, the polymer permeabilizes the outer and inner membranes; at higher polymer concentrations, permeabilization of the outer membrane is progressively diminished, while the inner membrane remains unaffected. Experiments with wild-type E. coli K12 show that the polymer blocks passage of solutes into the intermembrane space at high concentrations. Cell membrane integrity in E. coli K12 and S. aureus exhibits biphasic dependence on polymer concentration. Isothermal titration calorimetry indicates that the polymer associates with the negatively charged lipopolysaccharide of Gram-negative bacteria and with the lipoteichoic acid of Gram-positive bacteria. We propose that this polymer has two mechanisms of antibacterial action, one predominating at low concentrations of polymer and the other predominating at high concentrations.

Proregion anionic amino acids mediate inhibition of mouse pro‐α‐defensin bactericidal activity

Mouse Paneth cell α‐defensins are activated by proteolysis of inactive precursors, e.g., proCrp4 to bactericidal forms by the convertase MMP‐7. The activating step cleaves 24 residues, including 9 acidic amino acids that collectively block the membrane disruptive behavior of the Crp4 peptide (J Biol. Chem 281:28932–28943 2006). This inhibitory mechanism has been investigated further to identify whether specific cluster(s) of anionic amino acids in proCrp4(20–43) are preferentially responsible for blocking bactericidal activity and membrane disruption. To test this hypothesis, sets of Asp and Glu in proCrp4(20–43) were mutagenized to Gly. The recombinant proCrp4 variant peptides were expressed in E. coli, purified by HPLC to homogeneity and then assayed for differential effects of bactericidal inactivity. Generally, Gly for Glu/Asp replacements produced additive effects with respect to both bactericidal activity and membrane disruption of live E. coli ML35, regardless of their position. These findings support the conclusion that Asp and Glu residues contribute additively via intramolecular interactions to neutralize Crp4 membrane disruptive mechanisms. Supported by NIH grants DK044632 and AI059346

Membrane Insertion and Bilayer Perturbation by Antimicrobial Peptide CM15

Antimicrobial peptides (AMPs) are an important component of innate immunity and have generated considerable interest as a potential new class of antibiotic. The biological activity of AMPs is strongly influenced by peptide-membrane interactions; however, for many of these peptides the molecular details of how they disrupt and/or translocate across target membranes are not known. CM15 is a linear, synthetic hybrid AMP composed of the first seven residues of the cecropin A and residues 2–9 of the bee venom peptide mellitin. Previous studies have shown that upon membrane binding CM15 folds into an α-helix with its helical axis aligned parallel to the bilayer surface and have implicated the formation of 2.2–3.8 nm pores in its bactericidal activity. Here we report site-directed spin labeling electron paramagnetic resonance studies examining the behavior of CM15 analogs labeled with a methanethiosulfonate spin label (MTSL) and a brominated MTSL as a function of increasing peptide concentration and utilize phospholipid-analog spin labels to assess the effects of CM15 binding and accumulation on the physical properties of membrane lipids. We find that as the concentration of membrane-bound CM15 is increased the N-terminal domain of the peptide becomes more deeply immersed in the lipid bilayer. Only minimal changes are observed in the rotational dynamics of membrane lipids, and changes in lipid dynamics are confined primarily to near the membrane surface. However, the accumulation of membrane-bound CM15 dramatically increases accessibility of lipid-analog spin labels to the polar relaxation agent, nickel (II) ethylenediaminediacetate, suggesting an increased permeability of the membrane to polar solutes. These results are discussed in relation to the molecular mechanism of membrane disruption by CM15.

Induction of the cholesterol biosynthesis pathway in differentiated Caco-2 cells by the potato glycoalkaloid α-chaconine

Glycoalkaloids are naturally occurring toxins in potatoes, which at high levels may induce toxic effects in humans, mainly on the gastrointestinal tract by cell membrane disruption. In order to better understand the molecular mechanisms underlying glycoalkaloid toxicity, we examined the effects of α-chaconine on gene expression in the Caco-2 intestinal epithelial cell line using DNA microarrays. Caco-2 cells were exposed for 6 h to 10 μM α-chaconine in three independent experiments (randomized block design). The most prominent finding from our gene expression and pathway analyses was the upregulation of expression of several genes involved in cholesterol biosynthesis. This to some extent is in line with the literature-described mechanism of cell membrane disruption by glycoalkaloids. In addition, various growth factor signaling pathways were found to be significantly upregulated. This study is useful in understanding the mechanism(s) of α-chaconine toxicity, which may be extended to other potato glycoalkaloids more generally.

Mechanism of Supported Membrane Disruption by Antimicrobial Peptide Protegrin-1

While pore formation has been suggested as an important step in the membrane disruption process induced by antimicrobial peptides, membrane pore formation has never been directly visualized. We report on the dynamics of membrane disruption by antimicrobial peptide protegrin-1 (PG-1) on dimyristoyl-sn-glycero-phosphocholine-supported bilayer patches obtained via atomic force microscopy. The action of PG-1 is found to be concentration-dependent. At low PG-1 concentrations (1 < [PG-1] < 4 microg/mL), the peptide destabilizes the edge of the membrane to form fingerlike structures. At higher concentrations, PG-1 induces the formation of a sievelike nanoporous structure in the membrane. The highest degree of disruption is attained at concentrations >or=20 microg/mL, at which PG-1 disrupts the entire membrane, transforming it into stripelike structures with a well-defined and uniform stripe width. This first direct visualization of these membrane structural transformations helps elucidate the PG-1-induced membrane disruption mechanism.

Solid-State NMR Investigation of the Membrane-Disrupting Mechanism of Antimicrobial Peptides MSI-78 and MSI-594 Derived from Magainin 2 and Melittin

The mechanism of membrane interaction of two amphipathic antimicrobial peptides, MSI-78 and MSI-594, derived from magainin-2 and melittin, is presented. Both the peptides show excellent antimicrobial activity. The 8-anilinonaphthalene-1-sulfonic acid uptake experiment using Escherichia coli cells suggests that the outer membrane permeabilization is mainly due to electrostatic interactions. The interaction of MSI-78 and MSI-594 with lipid membranes was studied using 31P and 2H solid-state NMR, circular dichroism, and differential scanning calorimetry techniques. The binding of MSI-78 and MSI-594 to the lipid membrane is associated with a random coil to α-helix structural transition. MSI-78 and MSI-594 also induce the release of entrapped dye from POPC/POPG (3:1) vesicles. Measurement of the phase-transition temperature of peptide-DiPoPE dispersions shows that both MSI-78 and MSI-594 repress the lamellar-to-inverted hexagonal phase transition by inducing positive curvature strain. 15N NMR data suggest that both the peptides are oriented nearly perpendicular to the bilayer normal, which infers that the peptides most likely do not function via a barrel-stave mechanism of membrane-disruption. Data obtained from 31P NMR measurements using peptide-incorporated POPC and POPG oriented lamellar bilayers show a disorder in the orientation of lipids up to a peptide/lipid ratio of 1:20, and the formation of nonbilayer structures at peptide/lipid ratio >1:8. 2H-NMR experiments with selectively deuterated lipids reveal peptide-induced disorder in the methylene units of the lipid acyl chains. These results are discussed in light of lipid-peptide interactions leading to the disruption of membrane via either a carpet or a toroidal-type mechanism.

QSAR analysis of antimicrobial and haemolytic effects of cyclic cationic antimicrobial peptides derived from protegrin-1

In this paper we quantitatively analyse antimicrobial and haemolytic activities of porcine protegrin-1 (PG-1) mimetics—cyclic cationic peptides with β-hairpin fold synthesised by Robinson et al. [ Bioorg. Med. Chem. 2005, 13, 2055]. The presented QSAR models, which use molecular properties related to possible mechanisms of cell membrane disruption that can be easily calculated from available data on amino acids, rationalize the relationships between sequences and antimicrobial and haemolytic potencies of the cyclic peptides. The best models obtained by application of genetic function approximation algorithm correlate antimicrobial potencies to the peptide’s charge and amphipathicity index, while the haemolytic effect correlates well with the lipophilicity of residues forming the nonpolar face of the β-hairpin. The models permit selection of site-directed residue substitutions leading to simultaneous optimization of antimicrobial and haemolytic potencies. Examples of such residue substitutions in the nonpolar face of a symmetric cyclic β-hairpin PG-1 analogue with an ideal amphipathic structure are given.

Deletion of All Cysteines in Tachyplesin I Abolishes Hemolytic Activity and Retains Antimicrobial Activity and Lipopolysaccharide Selective Binding

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.

Induction of Morphological Changes in Model Lipid Membranes and the Mechanism of Membrane Disruption by a Large Scorpion-Derived Pore-Forming Peptide

The membrane disruption mechanism of pandinin 1 (pin1), an antimicrobial peptide isolated from the venom of the African scorpion, was studied using 31P, 13C, 1H solid-state and multidimensional solution-state NMR spectroscopy. A high-resolution NMR solution structure of pin1 showed that the two distinct α-helical regions move around the central hinge region, which contains Pro 19. 31P NMR spectra of lipid membrane in the presence of pin1, at various temperatures, showed that pin1 induces various lipid phase behaviors depending on the acyl chain length and charge of phospholipids. Notably, it was found that pin1 induced formation of the cubic phase in shorter lipid membranes above T m. Further, the 13C NMR spectra of pin1 labeled at Leu 28 under magic angle spinning (MAS) indicated that the motion of pin1 bound to the lipid bilayer was very slow, with a correlation time of the order of 10 −3 s. 31P NMR spectra of dispersions of four saturated phosphatidyl-cholines in the presence of three types of pin1 derivatives, [W4A, W6A, W15A]-pin1, pin1(1-18), and pin1(20-44), at various temperatures demonstrated that all three pin1 derivatives have a reduced ability to trigger the cubic phase. 13C chemical shift values for pin1(1-18) labeled at Val 3, Ala 10, or Ala 11 under static or slow MAS conditions indicate that pin1(1-18) rapidly rotates around the average helical axis, and the helical rods are inclined at ∼30° to the lipid long axis. 13C chemical shift values for pin1(20-44) labeled at Gly 25, Leu 28, or Ala 31 under static conditions indicate that pin1(20-44) may be isotropically tumbling. 1H MAS chemical shift measurements suggest that pin1 is located at the membrane-water interface approximately parallel to the bilayer surface. Solid-state NMR results correlated well with the observed biological activity of pin1 in red blood cells and bacteria.

Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A

Subtilosin A is an antimicrobial peptide produced by the soil bacterium Bacillus subtilis that possesses bactericidal activity against a diverse range of bacteria, including Listeria monocytogenes. Recent structural studies have found that subtilosin A is posttranslationally modified in a unique way, placing it in a new class of bacteriocins. In this study, in order to understand the mechanism of membrane-disruption by subtilosin A, the interaction of the peptide with model phospholipid bilayers is characterized using fluorescence, solid-state NMR and differential scanning calorimetry (DSC) experiments. Our results in this study show that subtilosin A interacts with the lipid head group region of bilayer membranes in a concentration dependent manner. Fluorescence experiments reveal the interaction of subtilosin A with small unilamellar vesicles (SUVs) composed of POPC, POPG and E. coli total lipids, and that at least one edge of the molecule is buried in membrane bilayers. At high concentrations, it induces leakage from SUVs of POPC and POPE/POPG (7:3) mixture. 15N solid-state NMR data suggests that the cyclic peptide is partially inserted into bilayers, which is in agreement with the fluorescence data. 31P and 2H NMR experiments and DSC data support the hypothesis that subtilosin A adopts a partially buried orientation in lipid bilayers, by showing that it induces a conformational change in the lipid headgroup and disordering in the hydrophobic region of bilayers. These results suggest that the lipid perturbation observed in this study may be one of the consequences of subtilosin A binding to lipid bilayers, which results in membrane permeabilization at high peptide concentrations.

A QSAR‐modeling perspective on cationic transfection lipids. 1. Predicting efficiency and understanding mechanisms

As gene therapy using viral vectors involves clinical risks, limited DNA-carrying capacity, and manufacturing problems, non-viral vectors, including cationic lipids, have been investigated. Unfortunately, these agents have significantly lower transfectional ability and, due to the complexity of the transfectional pathway, no general schemes exist for correlating cationic lipid chemistry with transfectional efficacy. Quantitative structure-activity relationship (QSAR) analyses were carried out on sets of routinely used, experimental, and unsuccessful cationic lipid vectors taken from the literature. This approach described the amphipathic character, basicity, headgroup size, lipophilicity and shape of cationic lipids using numerical parameters. Compounds were plotted onto various parameter diagrams, and correlations were sought between numerical parameters and transfectional efficiency. Transfectionally effective cationic lipids fell into restricted zones in various parameter spaces, indicating that amphipathic character, lipid shape and lipophilicity were generally significant factors, whilst basicity and headgroup size were only important for certain compounds. The data supported the general significance of membrane mixing followed by induction of membrane curvature, and the more limited role of osmotic shock, as mechanisms of membrane disruption. QSAR descriptions of effective lipids permitted detailed chemical guidelines for optimizing cationic lipid structure to be given. Limitations of the approach and models are discussed. QSAR modeling indicated that induction of membrane curvature and osmotic shock are important mechanisms for membrane disruption by cationic lipids. The models also allowed specification of chemically detailed guidelines for selection or design of optimal cationic lipids.