Fold-Unfold Transitions in the Selectivity and Mechanism of Action of the N-Terminal Fragment of the Bactericidal/Permeability-Increasing Protein (rBPI21)

Septic or endotoxic shock is a common cause of death in hospital intensive care units. In the last decade numerous antimicrobial peptides and proteins have been tested in the search for an efficient drug to treat this lethal disease. Now in phase III clinical trials, rBPI₂₁, a recombinant N-terminal fragment of the bactericidal/permeability-increasing protein (BPI), is a promising drug to reduce lesions caused by meningococcal sepsis. We correlated structural and stability data with functional information of rBPI₂₁ bound to both model systems of eukaryotic and bacterial membranes. On interaction with membranes, rBPI₂₁ loses its conformational stability, as studied by circular dichroism. This interaction of rBPI₂₁ at membrane level was higher in the presence of negatively charged phospholipid relatively to neutral ones, with higher partition coefficients (K_p), suggesting a preference for bacterial membranes over mammalian membranes. rBPI₂₁ binding to membranes is reinforced when its disulfide bond is broken due to conformational changes of the protein. This interaction is followed by liposome aggregation due to unfolding, which ensures protein aggregation, and interfacial localization of rBPI₂₁ in membranes, as studied by extensive quenching by acrylamide and 5-deoxylstearic acid and not by 16-deoxylstearic acid. An uncommon model of the selectivity and mechanism of action is proposed, where membrane induces unfolding of the antimicrobial protein, rBPI₂₁. The unfolding ensures protein aggregation, established by protein-protein interaction at membrane surface or between adjacent membranes covered by the unfolded protein. This protein aggregation step may lead to membrane perturbation.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Thermodynamic profiling of peptide membrane interactions by isothermal titration calorimetry: a search for pores and micelles

Antimicrobial peptides are known to interact strongly with negatively charged lipid membranes, initially by peripheral insertion of the peptide into the bilayer, which for some antimicrobial peptides will be followed by pore formation, and successive solubilization of the membranes resulting in mixed peptide-lipid micelles. We have investigated the mode of action of the antimicrobial peptide mastoparan-X using isothermal titration calorimetry (ITC) and cryo-transmission electron microscopy (cryo-TEM). The results show that mastoparan-X induces a range of structural transitions of POPC/POPG (3:1) lipid membranes at different peptide/lipid ratios. It has been established that ITC can be used as a fast method for localizing membrane transitions and when combined with DLS and cryo-TEM can elucidate structural changes, including the threshold for pore formation and micellation. Cryo-TEM was employed to confirm the structural changes associated with the thermodynamic transitions found by ITC. The pore-formation process has furthermore been investigated in detail and the thermodynamic parameters of pore formation have been characterized using a system-specific temperature where the enthalpy of peptide partitioning becomes zero (T_zero). This allows for an exclusive study of the pore-formation process. The use of ITC to find T_zero allows for characterization of the thermodynamic parameters of secondary processes on lipid membranes.     

Cell-Wall Interactions and the Selective Bacteriostatic Activity of a Miniature Oligo-Acyl-Lysyl

The oligo-acyl-lysyl, C_12(ω7)K-β₁₂, is comprised of only three Lys residues. Despite its small size, it exhibits potent bacteriostatic activity against Gram-positive bacteria, but it is ∼10-fold less potent against Gram-negative bacteria. We followed the interactions of C_12(ω7)K-β₁₂ from its initial contact with the bacterial surface across the cell wall down to the cytoplasmic membrane. Binding to anionic lipids, as well as to negatively charged LPS and LTA, occurs with very high affinity. The C_12(ω7)K-β₁₂ does not cross the outer membrane of Gram-negative bacteria; rather, it achieves its action by depositing on the LPS layer, promoting surface adhesion and blocking passage of solutes. In Gram-positive bacteria, the thick peptidoglycan layer containing LTA allows passage of C_12(ω7)K-β₁₂ and promotes its accumulation in the small periplasm. From that location it is then driven to the membrane by strong electrostatic interactions. Despite its high potency against Gram-positive bacteria, this agent is not capable of efficiently breaking down the permeability barrier of the cytoplasmic membrane or of reaching an intracellular target, as suggested by the fact that it does not interact with DNA.     

Aggregation of cateslytin beta-sheets on negatively charged lipids promotes rigid membrane domains. A new mode of action for antimicrobial peptides?

Cateslytin, a positively charged (5+) arginine-rich antimicrobial peptide (bCgA, RSMRLSFRARGYGFR), was chemically synthesized and studied against membranes that mimic bacterial or mammalian systems. Circular dichroism, polarized attenuated total reflection infrared spectroscopy, (1)H high-resolution MAS NMR, and (2)H and (31)P solid state NMR were used to follow the interaction from peptide and membrane points of view. Cateslytin, which is unstructured in solution, is converted into antiparallel beta-sheets that aggregate mainly flat at the surface of negatively charged bacterial mimetic membranes. Arginine residues are involved in the binding to negatively charged lipids. Following the interaction of the cateslytin peptide, rigid and thicker membrane domains enriched in negatively charged lipids are found. Much less interaction is detected with neutral mammalian model membranes, as reflected by only minor percentages of beta-sheets or helices in the peptide secondary structure. No membrane destruction was detected for both bacterial and mammalian model membranes. A molecular model is proposed in which zones of different rigidity and thickness bring about phase boundary defects that ultimately lead to permeability induction and peptide crossing through bacterial membranes.     

Interaction studies of novel cell selective antimicrobial peptides with model membranes and E. coli ATCC 11775

Cationic antimicrobial peptides (CAMPs) are novel candidates for drug development. Here we describe design of six short and potent CAMPs (SA-1 to SA-6) based on a minimalist template of 12 residues H+HHG+HH+HH+NH2 (where H: hydrophobic amino acid and +: charged hydrophilic amino acid). Designed peptides exhibit good antibacterial activity in micro molar concentration range (1-32 μg/ml) and rapid clearance of Gram-positive and Gram-negative bacterial strains at concentrations higher than MIC. For elucidating mode of action of designed peptides various biophysical studies including CD and Trp fluorescence were performed using model membranes. Further based on activity, selectivity and membrane bound structure; modes of action of Trp rich peptide SA-3 and template based peptide SA-4 were compared. Calcein dye leakage and transmission electron microscopic studies with model membranes exhibited selective membrane active mode of action for peptide SA-3 and SA-4. Extending our work from model membranes to intact E. coli ATCC 11775 in scanning electron micrographs we could visualize different patterns of surface perturbation caused by peptide SA-3 and SA-4. Further at low concentration rapid translocation of FITC-tagged peptide SA-3 into the cytoplasm of E. coli cells without concomitant membrane perturbation indicates involvement of intracellular targeting mechanism as an alternate mode of action as was also evidenced in DNA retardation assay. For peptide SA-4 concentration dependent translocation into the bacterial cytoplasm along with membrane perturbation was observed. Establishment of a non specific membrane lytic mode of action of these peptides makes them suitable candidates for drug development.     

A molecular dynamics study of the interaction between domain I-BAR of the IRSp53 protein and negatively charged membranes

The methods of computer simulation in all-atom and coarse-grained approximations have been used to study specific interactions of the isolated domain I-BAR of the actin-binding protein IRSp53 with model membranes containing neutral phospholipids and those including negatively charged PI(4,5)P₂ phospholipids. It has been shown that the I-BAR domain does not interact with neutral lipids but induces bending of the synthetic membrane rich in negatively charged phospholipids. Clustering of charged lipids on the surface of the membrane at the sites of its interaction with the protein has been observed. This indicates that the interaction of I-BAR with negatively charged lipids is of electrostatic and hydrophobic nature.     

Lipid binding specificity of bovine α-lactalbumin: A multidimensional approach

Many soluble proteins are known to interact with membranes in partially disordered states, and the mechanism and relevance of such interactions in cellular processes are beginning to be understood. Bovine α-lactalbumin (BLA) represents an excellent prototype for monitoring membrane interaction due to its conformational plasticity. In this work, we comprehensively monitored the interaction of apo-BLA with zwitterionic and negatively charged membranes utilizing a variety of approaches. We show that BLA preferentially binds to negatively charged membranes at acidic pH with higher binding affinity. This is supported by spectral changes observed with a potential-sensitive membrane probe and fluorescence anisotropy measurements of a hydrophobic probe. Our results show that BLA exhibits a molten globule conformation when bound to negatively charged membranes. We further show, using the parallax approach, that BLA penetrates the interior of negatively charged membranes, and tryptophan residues are localized at the membrane interface. Red edge excitation shift (REES) measurements reveal that the immediate environment of tryptophans in membrane-bound BLA is restricted, and the restriction is dependent on membrane lipid composition. We envision that understanding the mechanism of BLA–membrane interaction would help in bioengineering of α-lactalbumin, and to address the mechanism of tumoricidal and antimicrobial activities of BLA–oleic acid complex.     

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin’s antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.     

The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage

Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by Paenibacillus thiaminolyticus OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C₁₅ fatty acyl chain. The mechanism of action of paenibacterin against Escherichia coli and Staphylococcus aureus was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated the antimicrobial activity of paenibacterin against E. coli. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of E. coli and S. aureus. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

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.     

Binding of β-Amyloid (1-42) Peptide to Negatively Charged Phospholipid Membranes in the Liquid-Ordered State: Modeling and Experimental Studies

To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L_o) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L_o state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5–5 mol %) than higher (10–20 mol %) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L_o ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol % anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.     

Galleria mellonella native and analogue peptides Gm1 and ΔGm1. I) Biophysical characterization of the interaction mechanisms with bacterial model membranes

Natural occurring antimicrobial peptides (AMPs) are important components of the innate immune system of animals and plants. They are considered to be promising alternatives to conventional antibiotics. Here we present a comparative study of two synthetic peptides: Gm1, corresponding to the natural overall uncharged peptide from Galleria mellonella (Gm) and ΔGm1, a modified overall positively charged Gm1 variant. We have studied the interaction of the peptides with lipid membranes composed of different kinds of lipopolysaccharides (LPS) and dimyristoylphosphatidylglycerol (DMPG), in some cases also dimyristoylphosphatidylethanolamine (DMPE) as representative lipid components of Gram-negative bacterial membranes, by applying Fourier-transform infrared spectroscopy (FTIR), Förster resonance energy transfer spectroscopy (FRET), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). Gm1 generates a destabilizing effect on the gel to liquid crystalline phase transition of the acyl chains of the lipids, as deduced from a decrease in the phase transition temperature and enthalpy, suggesting a fluidization, whereas ΔGm1 led to the opposite behavior. Further, FTIR analysis of the functional groups of the lipids participating in the interaction with the peptides indicated a shift in the band position and intensity of the asymmetric PO₂⁻ stretching vibration originating from the lipid phosphate groups, a consequence of the sterical changes in the head group region. Interestingly, FRET spectroscopy showed a similar intercalation of both peptides into the DMPG and LPS, but much less into the DMPE membrane systems. These results are discussed in the light of a possible use of the peptides as antimicrobial and anti-endotoxin drugs.     

Mechanism of antibacterial action of a synthetic peptide with an Ala-peptoid residue based on the scorpion-derived antimicrobial peptide IsCT

A novel bacterial cell-selective antimicrobial peptide, IsCT-P (ILKKIWKPIKKLF-NH₂), was designed based on the scorpion-derived α-helical antimicrobial peptide, IsCT. Here, we investigated the effect of substituting Pro⁸ of IsCT-P with the Ala-peptoid residue (N-methylglycine) on the peptide’s structure and mechanism of action. Circular dichroism analysis revealed that the modified peptide, IsCT-a, has a much lower α-helicity than IsCT-P in membrane mimicking conditions, suggesting the peptoid residue provides much more structural flexibility than the proline residue. IsCT-a was also much less effective than IsCT-P at causing leakage of fluorescent dye entrapped within negatively charged vesicles and at dissipating the membrane potential of Staphylococcus aureus. Collectively, our results suggest that the antibacterial action of IsCT-a is due to the inhibition of intracellular targets rather than the disruption and depolarization of bacterial cell membranes.Shin Saeng Lim and Sang-Pil Yoon contributed equally to this work.     

Interactions of a synthetic Leu–Lys-rich antimicrobial peptide with phospholipid bilayers

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH₂) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC–dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d₅₄-DMPC) and mixed d₅₄-DMPC–DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC–DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by ³¹P NMR. However, no perturbations in the T ₁ relaxation nor the T ₂- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes).     

Selective acylation enhances membrane charge sensitivity of the antimicrobial peptide mastoparan-x

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N^α-terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other, more potent antimicrobial peptides in future studies.     

Calcium-activated membrane interaction of the islet amyloid polypeptide: implications in the pathogenesis of type II diabetes mellitus

The role played by Ca²⁺ ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca²⁺ ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca²⁺ ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca²⁺ ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of β-sheet rich hIAPP aggregates.     

Molecular design of antimicrobial peptides based on hemagglutinin fusion domain to combat antibiotic resistance in bacterial infection

Antimicrobial peptides are derived from the viral fusion domain of influenza virus hemagglutinin based on rational analysis of the intermolecular interaction between peptides and bacterial outer membrane. It is revealed that the isolated viral fusion domain is a negatively charged peptide HAfp^1‐23 that cannot effectively interact with the anionic membrane. Conversion of the native HAfp^1‐23 to a positively charged peptide HAfp^1‐23_KK by E11K/D19K mutation can promote the peptide‐membrane interaction substantially; this confers to the peptide a moderate antibacterial potency against antibiotic‐resistant bacterial strains. Cyclization of the linear peptide HAfp^1‐23_KK results in a cyclic peptide cHAfp^1‐23_KK, which can largely minimize entropy penalty upon the peptide‐membrane binding by pre‐stabilizing peptide hairpin configuration in solvent, where the linear peptide would incur in a considerable conformational change/folding from intrinsic disorder (in water) to the structured hairpin conformation (in lipid). As might be expected, the cyclization considerably improves peptide antibacterial activity with minimum inhibitory concentration of 67 and 34 μg/mL against multidrug‐resistant Pseudomonas aeruginosa and methicillin‐resistant Staphylococcus aureus, respectively.     

The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers.

The hexapeptide Ac-RRWWRF-NH2 has earlier been identified as a potent antimicrobial peptide by screening synthetic combinatorial hexapeptide libraries. In this study, it was found that this peptide had a large influence on the thermotropic phase behavior of model membranes containing the negatively charged headgroup phosphatidylglycerol, a major component of bacterial membranes. In contrast, differential scanning calorimetry showed that it had little effect on model membranes containing the zwitterionic phosphatidylcholine headgroup, the main component of erythrocyte membranes. This behavior is consistent with its biological activity and with its affinity to these membranes as determined by titration calorimetry, implying that peptide-lipid interactions play an important role in this process. The structure of this peptide bound to membrane-mimetic sodium dodecyl sulfate (SDS) and dodecylphosphocholine micelles has been determined using conventional two-dimensional nuclear magnetic resonance methods. It forms a marked amphipathic structure in SDS with its hydrophobic residues on one side of the structure and with the positively charged residues on the other side. This amphipathic structure may allow this peptide to penetrate deeper into the interfacial region of negatively charged membranes, leading to local membrane destabilization. Knowledge about the importance of electrostatic interactions of Arg and the role of Trp residues as a membrane interface anchor will provide insight into the future design of potent antimicrobial peptidomimetics.