Membrane Mimetic Environments Research Articles

Active Site Environment and Reactivity of Copper-Aβ in Membrane Mimetic SDS Micellar Environment.

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid β (Aβ) peptide in extracellular deposits generated upon proteolysis of Amyloid Precursor Protein (APP). While copper (Cu(II)) binds to Aβ in soluble oligomeric and aggregated forms, its interaction with membrane-bound Aβ remains elusive. Investigating these interactions is crucial for understanding AD pathogenesis. Here, utilizing SDS micelles as a simplified membrane mimic, we focus on elucidating the interplay between membrane-anchored Aβ and copper, given their pivotal roles in AD. We employed spectroscopic techniques including UV, CD, and EPR to characterize the active site of Cu-Aβ complexes. Our findings demonstrate that copper interacts with Aβ peptides in membrane-mimicking micellar environments similarly to aqueous buffer solutions. Cu-Aβ complexes in this medium also induce higher hydrogen peroxide (H2O2) production, potentially contributing to AD-related oxidative stress. Moreover, we observe an increased oxidation rate of neurotransmitter such as dopamine by Cu-Aβ complexes. These results enhance our understanding of Cu-Aβ interactions in AD pathology and offer insights into potential therapeutic interventions targeting this interaction.

Origin of Secondary Structure Transitions and Peptide Self-Assembly Propensity in Trifluoroethanol-Water Mixtures.

Membrane-peptide interactions are key to the formation of helical intermediates in the early stages of amyloidogenesis. Aqueous solutions of 2,2,2-trifluoroethanol (TFE) provide a membrane-mimetic environment capable of promoting and stabilizing local peptide interactions. Uperin 3.5 (U3.5), a 17-residue and amidated antimicrobial peptide, is unstructured in water but self-assembles into fibrils in the presence of salt. Secondary structure transitions linked to U3.5 self-assembly were investigated in TFE/water mixtures, in both the absence and presence of salt, to assess the role of membrane-peptide interactions on peptide self-assembly and amyloid formation. A 5-to-7-fold increase in fibril yield of U3.5 was observed at low TFE concentrations (10% TFE/water v/v) compared with physiological buffer but only in the presence of salt. No aggregation was observed in salt-free TFE/water mixtures. Circular dichroism spectra showed that partial helical structures, initially stabilized by TFE, transitioned to β-sheet-rich aggregates in a saline buffer. Molecular dynamics simulations confirmed that TFE and salt act synergistically to enhance peptide-peptide interactions, resulting in β-sheet-rich U3.5 oligomers at low TFE concentrations. Specifically, TFE stabilized amphipathic, helical intermediates, leading to increased peptide-peptide attraction through hydrophobic interactions. The presence of salt further enhanced the peptide-peptide interactions by screening positively charged residues. Thus, the study revealed the role of a membrane mimic in stabilizing helical intermediates on the pathway to amyloid formation in the antimicrobial U3.5 peptide.

Exploring the Efficacy of Peptides and Mimics against Influenza A Virus, Adenovirus, and Murine Norovirus.

The ongoing battle against viral pandemics continues, with the possibility of future outbreaks. The search for effective antiviral compounds that can combat a diverse range of viruses continues to be a focal point of research. This study investigated the efficacy of two natural antimicrobial peptides (AMPs) (lactoferricin and LL-37), two synthetic AMPs (melimine and Mel4), and nine AMP mimics (758, 1091, 1096, 1083, 610, NAPL, 3-BIPL, 4-BIPL, and Sau-22) against influenza A virus strains H1N1 and H3N2, human adenovirus 5 (HAdV-5), and murine norovirus 1 (MNV-1). These compounds were tested using virus pre-treatment, cell pre-treatment, or post-cell entry treatment assays, electron microscopy, and circular dichroism (CD), alongside evaluations of cytotoxicity against the host cells. After virus pre-treatment, the AMP mimics 610 and Sau-22 had relatively low IC50 values for influenza strains H1N1 (2.35 and 6.93 µM, respectively) and H3N2 (3.7 and 5.34 µM, respectively). Conversely, natural and synthetic AMPs were not active against these strains. For the non-enveloped viruses, the AMP Mel4 and mimic 1083 had moderate activity against HAdV-5 (Mel4 IC50 = 47.4 µM; 1083 IC50 = 47.2 µM), whereas all AMPs, but none of the mimics, were active against norovirus (LL-37 IC50 = 4.2 µM; lactoferricin IC50 = 23.18 µM; melimine IC50 = 4.8 µM; Mel4 IC50 = 8.6 µM). Transmission electron microscopy demonstrated that the mimics targeted the outer envelope of influenza viruses, while the AMPs targeted the capsid of non-enveloped viruses. CD showed that Mel4 adopted an α-helical structure in a membrane mimetic environment, but mimic 758 remained unstructured. The diverse activity against different virus groups is probably influenced by charge, hydrophobicity, size, and, in the case of natural and synthetic AMPs, their secondary structure. These findings underscore the potential of peptides and mimics as promising candidates for antiviral therapeutics against both enveloped and non-enveloped viruses.

Employment of mastoparan-like peptides to prevent Staphylococcus aureus associated with bovine mastitis.

Bovine mastitis is a frequent infection in lactating cattle, causing great economic losses. Staphylococcus aureus represents the main etiological agent, which causes recurrent and persistent intramammary infections because conventional antibiotics are ineffective against it. Mastoparan-like peptides are multifunctional molecules with broad antimicrobial potential, constituting an attractive alternative. Nevertheless, their toxicity to host cells has hindered their therapeutic application. Previously, our group engineered three mastoparan-L analogs, namely mastoparan-MO, mastoparan-R1, and [I5, R8] MP, to improve cell selectivity and potential. Here, we were interested in comparing the antibacterial efficacy of mastoparan-L and its analogs against bovine mastitis isolates of S. aureus strains, making a correlation with the physicochemical properties and structural arrangement changes promoted by the sequence modifications. As a result, the analog's hemolytic and/or antimicrobial activity was balanced. All the peptides displayed α-helical folding in hydrophobic and membrane-mimetic environments, as determined by circular dichroism. The peptide [I5, R8] MP stood out for its enhanced selectivity and antibacterial features related to mastoparan-L and the other derivatives. Biophysical approaches revealed that [I5, R8] MP rapidly depolarizes the bacterial membrane of S. aureus, causing cell death by subsequent membrane disruption. Our results demonstrated that the [I5, R8] MP peptide could be a starting point for the development of peptide-based drugs for the treatment of bovine mastitis, with the advantage of no residue in milk, which would help reduce the use of classical antibiotics.IMPORTANCEStaphylococcus aureus is a leading cause of mastitis, the world's most important dairy cattle disease. The multidrug resistance and zoonotic potential of S. aureus, besides the likelihood of antibiotic residues in milk, are of critical concern to public and animal health. Antimicrobial peptides offer a novel antimicrobial strategy. Here, we demonstrate that [I5, R8] MP is a potent and selective peptide, which acts on S. aureus by targeting the bacterial membrane. Therefore, understanding the physicochemical determinants and the modes of action of this class of antimicrobials opens novel prospects for peptide development with enhanced activities in the bovine mastitis context.

Relevance of amphiphilicity and helicity on the antibacterial action of a histatin 5-derived peptide.

Peptide dhvar4, derived from the active domain of our salivary peptide histatin 5, bears a Phe residue in the middle of its hydrophilic face when folded into an α-helix. We then synthesized an analog with this Phe replaced by Lys and two analogs preserving Phe but bearing two and three α-aminoisobutyric acid (Aib) residues to stabilize the helical structure. The aim of this design was to verify which of the two features is more favorable to the biological activity. We performed a conformational study by means of circular dichroism and nuclear magnetic resonance, made antibacterial tests, and assessed the stability of the peptides in human serum. We observed that amphiphilicity is more important than helix stability, provided a peptide can adopt a helical conformation in a membrane-mimetic environment.

Design, biological characteristics, and antibacterial mechanism of high therapeutic index antimicrobial peptides with PRRP as central axis.

As the important components of biological innate immunity, antimicrobial peptides (AMPs) were found in a variety of organisms including insects, plants, animals, bacteria, fungi, etc. However, high hemolytic activity, high toxicity, and poor stability of natural AMPs hinder serious their application as therapeutic agents. To overcome these problems, in this study we use PRRP as a central axis, and peptides were designed based on the sequence template XRRXXRXPRRPXRXXRRX-NH2, where X represents a hydrophobic amino acid like Phe (F), Ile (I), Val (V), and Leu (L). The designed peptides LR18, FR18, and IR18 showed effective antimicrobial activity against some Gram-positive bacteria and Gram-negative bacteria, low cytotoxicity to mammalian cells, and had a tendency to form α-helical structures in membrane-mimetic environments. Among them, peptide LR18 (X: L) showed the highest geometric mean average treatment index (GMTI = 42.7) against Gram-negative bacteria, and FR18 (X: L) showed the highest GMTI (22.86) against Gram-positive bacteria. LR18 and FR18 also showed better salt, temperature, pH, and trypsin stability. LR18 and FR18 exert their antimicrobial effects mainly through destroying bacteria cell membrane. Briefly, peptide LR18 and FR18 have the potential to serve as a therapeutic agent to reduce antibiotic resistance owing to its high therapeutic index and great stability.

Antimicrobial peptide 2K4L disrupts the membrane of multidrug-resistant Acinetobacter baumannii and protects mice against sepsis.

Antimicrobial peptides represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. 2K4L is a rationally-designed analog of a short peptide temporin-1CEc, a natural peptide isolated and purified from the skin secretions of the Chinese brown frog Rana chensinensis by substituting amino acid residues. 2K4L adopt an α-helical confirm in a membrane-mimetic environment and displayed an improved and broad-spectrum antibacterial activity against sensitive and multidrug-resistant Gram-negative and Gram-positive bacterial strains. Here, the action mechanism of 2K4L on multidrug resistant Acinetobacter baumannii (MRAB) and protection on MRAB-infected mice was investigated. The results demonstrated high bactericidal activity of 2K4L against both a multidrug resistant A. baumannii 0227 strain (MRAB 0227) and a sensitive A. baumannii strain (AB 22934), indicating a potential therapeutic advantage of this peptide. Strong positively-charged residues significantly promoted the electrostatic interaction on 2K4L with lipopolysaccharides (LPS) of the bacterial outer membrane. High hydrophobicity and an α-helical confirm endowed 2K4L remarkably increase the permeability of A. baumannii cytoplasmic membrane by depolarization of membrane potential and disruption of membrane integration, as well as leakage of fluorescein from the liposomes. Additionally, 2K4L at low concentrations inhibited biofilm formation and degraded mature 1-day-old MRAB 0227 biofilms by reducing the expression of biofilm-related genes. In an invasive A. baumannii infection model, 2K4L enhanced the survival of sepsis mice and decreased the production of the proinflammatory cytokines downregulating the phosphorylation level of signaling protein in MAPK and NF-κB signaling pathways, indicating that 2K4L represents a novel therapeutic antibiotic candidate against invasive multidrug-resistant bacterial strain infections.

Tree Frog-Derived Cathelicidin Protects Mice against Bacterial Infection through Its Antimicrobial and Anti-Inflammatory Activities and Regulatory Effect on Phagocytes.

Due to excessive use or abuse in the food industry, agriculture, and medicine, many pathogens are developing resistance against conventional antibiotics. Antimicrobial peptides (AMPs) hold promise as effective therapeutic options for the treatment of bacterial infections. Herein, a novel cathelicidin antimicrobial peptide (Zs-CATH) was identified from the tree frog Zhangixalus smaragdinus. Zs-CATH mainly adopted an amphipathic β-sheet structure in a membrane-mimetic environment. It showed broad-spectrum antibacterial activity against Gram-positive and Gram-negative bacteria in vitro and significantly protected mice from lethal infections induced by Gram-negative bacteria Escherichia coli ATCC 25922 or Gram-positive bacteria Staphylococcus aureus ATCC 25923 in vivo. In addition, Zs-CATH exerted a strong anti-inflammatory effect by neutralizing lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and promoting macrophage M2 polarization, thus inhibiting the secretion of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) and enhancing the production of M2 macrophage markers IL-10, IL-4, and CD206. The MAPK and NF-κB inflammatory signaling pathways and transcriptional activator 6 (STAT6) were involved in this effect. In mice, Zs-CATH rapidly recruited neutrophils and monocytes/macrophages to the abdominal cavity but not T and B lymphocytes. Zs-CATH did not exhibit a direct chemoattractant effect on phagocytes but significantly promoted phagocyte migration in the presence of macrophages. Zs-CATH stimulated macrophages to secrete chemokines CXCL1, CXCL2, and CCL2, which mediated the recruitment of phagocytes. Furthermore, Zs-CATH promoted the production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs), which are oxygen-dependent and oxygen-independent mechanisms of the microbicidal activity of neutrophils, respectively. Zs-CATH exhibited no toxic side effects on mammalian cells and mice. These findings show that in addition to direct antibacterial activity, Zs-CATH also possesses the ability to modulate immune and inflammatory processes during bacterial infection, showing potential for development as anti-infective and/or anti-inflammatory agents.

Effect of central PxxP motif in amphipathic alpha-helical peptides on antimicrobial activity and mode of action

Amphipathic α-helical peptides (AHPs) have shown potential as a therapeutic approach against multi-drug-resistant bacterial infections due to their broad-spectrum antimicrobial activity by disrupting bacterial membranes. However, their nonspecific interactions with membranes often result in cytotoxicity toward mammalian cells. Previous studies have shown that a PxxP motif near the middle of cathelicidin-derived antimicrobial peptides contributes to potent and selective antibacterial activity. In this study, we compared KL18 with KL-PxxP to examine the effects of the central PxxP motif in AHPs on their structure, antibiotic activity, and mode of action. In a membrane-mimetic environment, we observed that KL18 had a much higher helical content compared to KL-PxxP. In aqueous buffer, KL18 adopted a highly ordered α-helical conformation, while KL-PxxP exhibited a disordered conformation. We found that KL-PxxP exhibited 4–16 times higher antibacterial activity than KL18 and significantly reduced the hemolytic activity. These findings suggest that the dynamic conformational behaviors caused by the central PxxP motif conferred the antibacterial selectivity of AHPs. Additionally, KL-PxxP showed strong binding to anionic liposomes and weak binding to zwitterionic liposomes, explaining its selectivity for bacteria over mammalian cells. Despite having a low ability to dissipate the bacterial membrane potential, KL-PxxP translocated efficiently across lipid membranes. Therefore, we propose that the central PxxP motif in AHPs provides dynamic conformational behavior in aqueous and membrane-mimetic environments, enhances binding to anionic membranes, and facilitates translocation across lipid bilayers, resulting in improved antibacterial potency and selectivity. Understanding the unique structural characteristics and functional roles of the PxxP motif in the antimicrobial mechanism of action holds great potential for advancing the development of novel peptide antibiotics.

Insight on the mechanism of hexameric Pseudin-4 against bacterial membrane-mimetic environment.

As an alternative to antibiotics, Antimicrobial Peptides (AMPs) possess unique properties including cationic, amphipathic and their abundance in nature, but the exact characteristics of AMPs against bacterial membranes are still undetermined. To estimate the structural stability and functional activity of AMPs, the Pseudin AMPs (Pse-1, Pse-2, Pse-3, and Pse-4) from Hylid frog species, Pseudis paradoxa, an abundantly discovered source for AMPs were examined. We studied the intra-peptide interactions and thermal denaturation stability of peptides, as well as the geometrical parameters and secondary structure profiles of their conformational trajectories. On this basis, the peptides were screened out and the highly stable peptide, Pse-4 was subjected to membrane simulation in order to observe the changes in membrane curvature formed by Pse-4 insertion. Monomeric Pse-4 was found to initiate the membrane disruption; however, a stable multimeric form of Pse-4 might be competent to counterbalance the helix-coil transition and to resist the hydrophobic membrane environment. Eventually, hexameric Pse-4 on membrane simulation exhibited the hydrogen bond formation with E. coli bacterial membrane and thereby, leading to the formation of membrane spanning pore that allowed the entry of excess water molecules into the membrane shell, thus causing membrane deformation. Our report points out the mechanism of Pse-4 peptide against the bacterial membrane for the first time. Relatively, Pse-4 works on the barrel stave model against E. coli bacterial membrane; hence it might act as a good therapeutic scaffold in the treatment of multi-drug resistant bacterial strains.

Heme-Aβ in SDS micellar environment: Active site environment and reactivity

Alzheimer's disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder that causes brain cell death. Oxidative stress derived from the accumulation of redox cofactors like heme in amyloid plaques originating from amyloid β (Aβ) peptides has been implicated in the pathogenesis of AD. In the past our group has studied the interactions and reactivities of heme with soluble oligomeric and aggregated forms of Aβ. In this manuscript we report the interaction of heme with Aβ that remains membrane bound using membrane mimetic SDS (sodium dodecyl sulfate) micellar medium. Employing different spectroscopic techniques viz. circular dichroism (CD), absorption (UV–Vis), electron paramagnetic resonance (EPR) and resonance Raman (rR) we find that Aβ binds heme using one of its three His (preferentially His13) in SDS micellar medium. We also find that Arg5 is an essential distal residue responsible for higher peroxidase activity of heme bound Aβ in this membrane mimetic environment than free heme. This peroxidase activity exerted by even membrane bound heme-Aβ can potentially be more detrimental as the active site remains close to membranes and can hence oxidise the lipid bilayer of the neuronal cell, which can induce cell apoptosis. Thus, heme-Aβ in solution as well as in membrane-bound form are detrimental.

Temperature-Induced Effects on the Structure of Gramicidin S.

We report on the structure of Gramicidin S (GS) in a model membrane mimetic environment represented by the amphipathic solvent 1-octanol using one-dimensional (1D) and two-dimensional (2D) IR spectroscopy. To explore potential structural changes of GS, we also performed a series of spectroscopic measurements at differing temperatures. By analyzing the amide I band and using 2D-IR spectral changes, results could be associated to the disruption of aggregates/oligomers, as well as structural and conformational changes happening in the concentrated solution of GS. The ability of 2D-IR to enable differentiation in melting transitions of oligomerized GS structures is attributed to the sensitivity of the technique to vibrational coupling. Two melting transition temperatures were identified; at Tm1 in the range 41-47 °C where the GS aggregates/oligomers disassemble and at Tm2 = 57 ± 2 °C where there is significant change involving GS β-sheet-type hydrogen bonds, whereby it is proposed that there is loss of interpeptide hydrogen bonds and we are left with mainly intrapeptide β-sheet and β-turn hydrogen bonds of the smaller oligomers. Further analysis with quantum mechanical/molecular mechanics (QM/MM) simulations and second derivative results highlighted the participation of active GS side chains. Ultimately, this work contributes toward understanding the GS structure and the formulation of GS analogues with improved bioactivity.

Elucidating the mechanisms underlying the action of kenaf seed peptides mixture against gram-positive and gram-negative bacteria and its efficacy in whole milk preservation

The present study determined the in-situ antibacterial effect of cationic kenaf seed peptides mixture (KSPM) in whole milk and further comprehensively elucidated the mechanisms underlying the bactericidal action against gram-positive and gram-negative bacteria. The application experiments in artificially contaminated milk showed that KSPM at 0.5 and 1 mg/mL displayed a stable and strong bactericidal activity within 1–6 h of treatment and lasted up to two weeks storage at room and refrigeration conditions. The in-vitro mechanistic study showed that the KSPM underwent conformational changes from random coil to α-helix and β-sheet in the membrane-mimetic environment. The KSPM exerted its inhibitory effect in a concentration and time-dependent manner through cell membrane permeabilisation associated with membrane-disrupting effects, which is characterised by the cellular material leakage. The in-silico study revealed that the peptides efficiently interacted with bacterial peptidoglycan via hydrogen bonding, electrostatic interactions and hydrophobic forces. The peptide RALPSEHNK interacted with gram-positive and gram-negative bacterial phospholipid-phosphate head group mainly through electrostatic interaction. The current work indicated that KSPM exerts its bactericidal effect by membrane-targeted mechanism and it would be a promising natural candidate to control bacterial contamination in milk and dairy-based products.

Circular Dichroism Study of Orexin B under Oxidative Stress Conditions.

The neuropeptides orexin A and B regulate various vital functions of the body, such as sleep/wake states, metabolism, and energy homeostasis. A loss of their physiological activity, with reduced ability to recognize their receptors, is suspected to be associated with oxidative stress conditions. These are related to excessive presence of reactive oxygen and nitrogen species, as well as of reactive lipoxidation byproducts. With the aim of evaluating the effects of oxidative stress on the secondary structure of orexin peptides, orexin B was synthesized and characterized by circular dichroism spectroscopy under different conditions. In aqueous solution it presents an unordered conformation, while in a membrane mimetic environment it assumes a helical structure. The effects of oxidative stress were evaluated exposing it to both oxygen and nitrogen radicals as well as to lipoxidation byproducts. The results showed that ROS, but not NRS, induced appreciable conformational changes, and only in the membrane mimetic environment. Lipoxidation byproducts, instead, led to secondary structure modifications much more evident than those induced by the direct action of ROS and RNS, and in both analyzed media. Additionally, MALDI-TOF analyses detected mass variations in the peptide attributable to oxidation of the C-terminal Met residue and deamination of asparagine in the Asn-His sequence. Taken together, all these data seem to confirm the involvement of oxidative processes in dysfunctions of the orexinergic system.

The cationic nature of lysine-rich segments modulates the structural and biochemical properties of wild potato FSK3 dehydrin

Dehydrins are hydrophilic stress-induced proteins that are thought to protect cellular machinery from the adverse effect of dehydration caused by low temperature, drought, or salinity. In the previous study, acidic FSK3 dehydrin DHN24 from Solanum sogarandinum was found to accumulate at multiple sites in phloem cells in response to cold treatment. This study investigated the biochemical and structural properties of recombinant DHN24. It was shown that the overexpression of DHN24 in Escherichia coli led to the inhibition of bacterial growth. The purified DHN24 was found to protect lactate dehydrogenase from freeze-induced denaturation. Circular dichroism (CD) analysis showed that DHN24 was disordered in aqueous solutions, but adopted α-helical conformation in a membrane-mimetic environment using sodium dodecyl sulfate micelles. DHN24 also interacted with anionic phosphatidic acid (PA). DHN24 contains four lysine-rich regions including three K-segments and a region upstream of the S-segment. The role of their local cationic nature is unknown. These segments are predicted to form helical structures. The CD analysis of mutant proteins in the membrane-mimetic environment matched these predictions most closely, revealing that the positively charged lysine residues in these regions promoted disorder-to-order transitions. Moreover, the inhibition of bacterial growth and interactions with PA were regulated by the local cationic nature of DHN24, while no such regulation was observed for its cryoprotective activity. The importance of the positive charge of the lysine-rich segments and disordered structure for DHN24 activity is discussed in relation to its possible biological function.

10-Residue MyD88-Peptide Adopts β-Sheet Structure, Self-Assembles, Binds to Lipopolysaccharides, and Rescues Mice from Endotoxin-Mediated Lung-Infection and Death.

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine-valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

Lipid membrane mimetics and oligomerization tune functional properties of proteorhodopsin

The functional properties of proteorhodopsin (PR) have been found to be strongly modulated by oligomeric distributions and lipid membrane mimetics. This study aims to distinguish and explain their effects by investigating how oligomer formation impacts PR’s function of proton transport in lipid-based membrane mimetic environments. We find that PR forms stable hexamers and pentamers in both E. coli membranes and synthetic liposomes. Compared with the monomers, the photocycle kinetics of PR oligomers is ∼2 and ∼4.5 times slower for transitions between the K and M and the M and N photointermediates, respectively, indicating that oligomerization significantly slows PR’s rate of proton transport in liposomes. In contrast, the apparent pKa of the key proton acceptor residue D97 (pKaD97) of liposome-embedded PR persists at 6.2–6.6, regardless of cross-protomer modulation of D97, suggesting that the liposome environment helps maintain PR’s functional activity at neutral pH. By comparison, when extracted directly from E. coli membranes into styrene-maleic acid lipid particles, the pKaD97 of monomer-enriched E50Q PR drastically increases to 8.9, implying that there is a very low active PR population at neutral pH to engage in PR’s photocycle. These findings demonstrate that oligomerization impacts PR’s photocycle kinetics, while lipid-based membrane mimetics strongly affect PR’s active population via different mechanisms.

The synthetic antimicrobial peptide IKR18 displays anti-infectious properties in Galleria mellonella in vivo model

Antimicrobial peptides (AMPs) are promising tools for developing new antibiotics. We described the design of IKR18, an AMP designed with the aid of computational tools. IKR18 showed antimicrobial activity against Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). CD studies revealed that IKR18 assumes an alpha-helical structure in the membrane-mimetic environment. The action mechanism IKR18 involves damage to the bacteria membrane, as demonstrated by Sytox green uptake. Furthermore, IKR18 displayed synergic and additive effects in combination with antibiotics ciprofloxacin and vancomycin. The peptide showed anti-biofilm activity in concentration and efficiency compared with commercial antibiotics, involving the direct death of bacteria, as confirmed by scanning electron microscopy. The anti-infective activity of IKR18 was demonstrated in the Galleria mellonella model infected with S. aureus, MRSA, and Acinetobacter baumannii. The novel bioinspired peptide, IKR18, proved to be effective in the control of bacterial infection, opening opportunities for the development of further assays, including preclinical models.

Three-Dimensional Structure of the Antimicrobial Peptide Cecropin P1 in Dodecylphosphocholine Micelles and the Role of the C-Terminal Residues.

Cecropin P1 (CP1) isolated from a large roundworm Ascaris suum, which is found in pig intestines, has been extensively studied as a model antimicrobial peptide (AMP). However, despite being a model AMP, its antibacterial mechanism is not well understood, particularly the function of its C-terminus. By using an Escherichia coli overexpression system with calmodulin as a fusion partner, we succeeded in the mass expression of recombinant peptides, avoiding toxicity to the host and degradation of CP1. The structure of the recombinant 15N- and 13C-labeled CP1 and its C-terminus truncated analogue in dodecylphosphocholine (DPC) micelles was determined by NMR. In this membrane-mimetic environment, CP1 formed an α-helix for almost its entire length, except for a short region at the C-terminus, and there was no evidence of a hinge, which is considered important for the expression of activity in other cecropins. Several NMR analyses showed that the entire length of CP1 was protected from water by micelles. Since the loss of the C-terminus of the analogue had little effect on the NMR structure or its interaction with the micelle, we investigated another role of the C-terminus of CP1 in its antimicrobial activity. The results showed that the C-terminal region affected the DNA-binding capacity of CP1, and this mechanism of action was also newly suggested that it contributed to the antimicrobial activity of CP1.

Anionic lipids induce a fold-unfold transition in the membrane-translocating Engrailed homeodomain

Homeoprotein transcription factors have the property of interacting with membranes through their DNA-binding homeodomain, which is involved in unconventional internalization and secretion. Both processes depend on membrane-translocating events but their detailed molecular mechanisms are still poorly understood. We have previously characterized the conformational properties of Engrailed 2 homeodomain (EnHD) in aqueous solution and in micelles as membrane-mimetic environments. In the present study, we used small isotropic lipid bicelles as a more relevant membrane-mimetic model to characterize the membrane-bound state of EnHD. We show that lipid bicelles, in contrast to micelles, adequately reproduce the requirement of anionic lipids in the membrane binding and conformational transition of EnHD. The fold-unfold transition of EnHD induced by anionic lipids was characterized by NMR using 1H, 13C, 15N chemical shifts, nuclear Overhauser effects, residual dipolar couplings, intramolecular and intermolecular paramagnetic relaxation enhancements induced by site-directed spin-label or paramagnetic lipid probe, respectively. A global unpacking of EnHD helices is observed leading to a loss of the native fold. However, near-native propensities of EnHD backbone conformation are maintained in membrane environment, including not only the three helices but also the turn connecting helices H2 and H3. NMR and coarse-grained molecular dynamics simulations reveal that the EnHD adopts a shallow insertion in the membrane, with the three helices oriented parallel to the membrane. EnHD explores extended conformations and closed U-shaped conformations, which are stabilized by anionic lipid recruitment.