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One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as −2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function.

The increasing number of multidrug resistant bacteria to available antibiotics is a growing problem worldwide. One major strategy for resistance and an important reason for failure of therapy in the clinic is biofilm formation. To cope with unfavorable surroundings many bacteria live as biofilms, sessile micro-colonies adherent to surfaces that secrete an extracellular polymeric substance. An attractive alternative to conventional antibiotics are antimicrobial peptides (AMPs), innate immune system molecules that target the bacterial cytoplasmic membrane, causing membrane disruption and cell death. AMPs biophysical properties are extensively studied regarding planktonic bacteria but much less so regarding biofilm formation. By designing and synthesizing a series of model peptides that share the same amino acids composition but differ in their biophysical properties, we investigated how different steps of biofilm formation are affected by the AMP's features. We modified the peptides characteristics using different approaches of charge segregation and amino acids D enantiomers. We used Pseudomonas aeruginosa, an opportunistic Gram-negative bacteria, which is a leading cause of severe pulmonary infections in cystic fibrosis patients and medical device contamination. Our work demonstrates that the peptides combat biofilm at different stages of its formation and maintenance: (1) killing bacteria at their planktonic stage (2) preventing bacterial adhesion to biomaterials and (3) degrading pre-formed biofilm. We show that substitution of L-to-D amino acids alters the peptides biophysical properties and improves their activity against each stage in the biofilm life cycle. By investigating which biophysical properties are essential for anti-biofilm activity we also discovered new mechanisms of peptides activity.

Macrophages are part of the immune system and play critical role in the host defense and tissue homeostasis ingesting a dead tissue and fighting invading pathogens. Exploring interactions of anticancer therapies with macrophages as part of innate immunity is important both for biomedical researches and applications and can help to combine various approaches with immunotherapy. In this study we evaluated the influence of anticancer drug colchicine on the disorder and complexity of the macrophage membrane from atomic force microscope (AFM) images. Fixed and dried mouse RAW 264.7 macrophage membranes were imaged with AFM operating in Peak Force mode. The disorder of the membrane was characterized by entropy and complexity - by fractal dimension. These parameters were calculated for a set of AFM images of untreated macrophages and macrophages pretreated with anticancer drug colchicine, using three different methods. Processing of AFM images and calculations were done with custom MATLAB code. We show that colchicine treatment yields entropy increase and therefore produces higher disorder of the macrophage membrane. Furthermore the membrane complexity is reduced demonstrating lower fractal dimension. In addition we studied also changes in the macrophage membrane disorder and complexity produced by microtubule stabilizing agent taxol, having opposite to the colchicine (inhibiting the microtubule polymerization) effect. These results demonstrate at the level of single cell that anticancer drug colchicine affects macrophage membrane structures producing more disordered state. Finally we discuss some possible consequences of this more disordered state on the macrophage activity.

Lipopeptides such as polymyxins, octapeptins or daptomycin often show an increased activity against bacteria as compared to their non-acylated analogue. Thus, we have studied N-acylated synthetic peptides derived from a fragment of human lactoferrin (LF-11) to elucidate the interaction of these peptides with Gram-negative and Gram-positive bacteria and membrane mimetic systems using various biophysical and biological methods.Calorimetric studies on liposomes composed of phosphatidylglycerol revealed that the parent peptide induced a phase separation into peptide-enriched and -poor domains, which however consist of a similar domain size as calculated by the cooperative units. In contrast, at the same lipid-to-peptide molar ratio (25:1) the N-acylated derivatives strongly broadened the phase transition range and lowered markedly the main transition temperature. This is indicative for rather small and inhomogeneous domains, which will result in large line defects increasing membrane permeability as observed in intact bacteria. Membrane destabilization of E. coli and S. aureus induced by the peptides was monitored by using the membrane-potential-sensitive dye DiIC1 and the extent of membrane damage caused by the peptides by the cationic dye SYTOX green, which cannot enter intact cells unless its membrane is disrupted by external compounds. In both assays the N-acylated peptides showed a dramatic increase of fluorescence indicating massive membrane damage. This is supported by electron micrographs, which clearly showed a loss of cytoplasmic content and membrane rupture in the presence of the N-acylated peptide. Nevertheless, the extent of cell membrane rupture does not necessarily strongly correlate with the MIC-value of the peptides emphasizing the different mode of interaction of (non)-acylated peptides, which in part may be related to different degree of interaction with cell membrane/wall components such as lipopolysaccharides and lipoteichoic acid.Acknowledgement to EC-projects “ANEPID” and “BIOCONTROL”

Human immunodeficiency virus (HIV-1) fusion is mediated by the gp41 subunit of the envelope glycoprotein (ENV). Folding into the post-fusion conformation is apparently the rate limiting step for the fusion reaction and it enables inhibition of the fusion process. This is demonstrated by the inhibitory capability of N- or C-peptides derived from the N-or C-terminal heptad repeat (NHR or CHR) regions, respectively. These peptides have been shown to bind their endogenous counterparts thereby preventing progression into the post-fusion conformation and arresting fusion. Since N-peptides tend to aggregate, C-peptides are more potent fusion inhibitors, and therefore, attracted most therapeutic effort and research. Here we show that a short peptide derived from the classical NHR region is highly potent inhibitor only when linked to a fatty acid at the N- and not the C-terminus. To our knowledge, this is the shortest potent inhibitory peptide derived from the NHR region. Using biophysical and cellular approaches, we: (i) revealed that its mode of action is altered from other classical N-peptide, and (ii) suggest a new motif in the NHR involved in stabilization of the post-fusion conformation. Besides shedding light on the mechanism of HIV-cell fusion, the similarity between the ENV of lentiviruses provides a new approach for designing short inhibitors from the NHR region of other viruses as well.

The molecular mechanism, by which bacteria sense antimicrobial peptides (AMPs) that promote its virulence is partially known. Bacteria are capable of changing the expression of virulence genes essential to survival and replication, by sensing changes in their microenvironment within the tissues of their host. As a consequence they have the potential to develop resistance to AMPs. In the case of Salmonella typhimurium, some of the virulence genes are controlled by the two component regulatory system, PhoP/PhoQ. The sensor protein of this system, PhoQ, is directly activated by antimicrobial peptides (AMPs). PhoQ phosporylates and activates PhoP, a transcriptional regulatory protein, which in turn activates or represses over 40 different genes. The activation of these genes was found to be essential to the survival of these pathogenic bacteria within the host macrophages. However, it is not yet clear whether this mechanism is shared by AMPs in general, or it requires specific biophysical properties for AMPs such as secondary structure, amino acid composition or specific sequence. Our studies reveal that changing the biophysical properties of a peptide that can induce resistance, such as incorporation of D-amino acids, can improve the peptides activity against Salmonella typhimurium probably by affecting the two component system. Studes along this line suggest that such peptide modifications can be used in order to overcome the inducible resistance of Salmonella typhimurium.

The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/ phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of > 2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 +/- 3 peptides. Interestingly, less leakage occurred at 43 degrees C, than at 30 degrees C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30 degrees C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin.

Toll Like Receptors (TLRs) are major facilitators of the innate immune response. Although the structure and function of TLRs are well studied, the role of their transmembrane domain has not been fully understood. Here we show that peptides based on the TM domain of TLR2 and TLR1/6 specifically inhibit TLR2 activation. These peptides inhibited TNFα secretion by mouse macrophages after lipotechoic acid (LTA, the cell wall of Gram-positive bacteria) activation, while no effect was seen on TLR4 activation by lipopolysacharide (LPS, the cell wall of Gram-negative bacteria). FRET analysis and self assembly assays revealed that these TM peptides are capable of interacting with each other, favoring hetero- rather than homo-dimerisation. Finally, a selected TLR2 TM peptide was able to rescue up to 60% of mice affected by an otherwise lethal acute systemic inflammation driven by hyperactivation of TLR2.

Membrane and antimicrobial properties of three short Arg-rich peptides containing the same amino acid composition but different sequences were determined in this study. These peptides, PFWRIRIRR-amide (PR-9), RRPFWIIRR-amide (RR-9) and PRFRWRIRI-amide (PI-9), all exhibit the ability to induce segregation of the anionic lipids from the zwitterionic lipids, as shown by changes in the phase transition properties of lipid mixtures detected by differential scanning calorimetry and also by freeze fracture electron microscopy. The Minimal Inhibitory Concentration (MIC) of these three peptides against several strains of Gram positive bacteria correlated well with the lipid composition of the bacterial membrane. The lower activity of these three peptides against Gram negative bacteria, particularly PI-9, could be explained by the interactions of these peptides with LPS as shown by isothermal titration calorimetry. The promotion of lipid domains by PR-9 as well as by a cathelicidin fragment, KR-12 that had previously been shown to induce lipid phase segregation, was directly visualized using freeze fracture electron microscopy. This work shows the insensitivity of phase segregation to the specific arrangement of the cationic charges in the sequence of these small cationic peptides as well as being independent of their tendency to form different secondary structures.

The initial step of HIV infection is fusion between the viral and target cell membranes. Fusion is mediated by the HIV gp41 protein and its N-terminal “fusion peptide” (FP) which binds to target cell membranes. Shorter constructs of gp41 that contain the FP usually catalyze vesicle fusion and such fusion at physiological pH was measured for three different gp41 constructs which differed in their numbers of N-terminal gp41 residues. “FP34” and “N70” were respectively models of the FP and “pre-hairpin intermediate” gp41 conformation while “FP-hairpin” was a model of the final “six-helix-bundle” gp41 structure. N70 induced rapid fusion, FP34 induced moderate fusion, and FP-hairpin induced no fusion and even arrested fusion induced by FP34. The data therefore suggest that the six-helix bundle conformation stops membrane fusion. In related work, solid-state nuclear magnetic resonance measurements probed the membrane locations of three different FP constructs with very different fusion rates. There was a positive correlation between fusion rate and depth of membrane insertion for the FP in either helical or β strand conformation. The key determinant of fusion rate may therefore be FP membrane location rather than conformation.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The hydropathy plot of ROMK1, an inwardly rectifying K+ channel, suggests that the channel contains two transmembrane domains (M1 and M2) and a linker between them with significant homology to the H5 pore region of voltage-gated K+ channels. To gain structural information on the pore region of the ROMK1 channel, we used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability of the putative membranous domains of the ROMK1 channel to self-assemble and coassemble within lipid membranes. Circular dichroism (CD) spectroscopy revealed that M1 and M2 adopt high alpha-helical structures in egg phosphatidylcholine small unilamellar vesicles and 40% trifluoroethanol (TFE)/water, whereas H5 is not alpha-helical in either egg phosphatidylcholine small unilamellar vesicles or 40% TFE/water. Binding experiments with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled peptide demonstrated that all of the peptides bind to zwitterionic phospholipid membranes with partition coefficients on the order of 10(5) M-1. Tryptophan quenching experiments using brominated phospholipids revealed that M1 is dipped into the hydrophobic core of the membrane. Resonance energy transfer (RET) measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that H5 and M2 can self-associate in their membrane-bound state, but M1 cannot. Moreover, the membrane-associated nonhelical H5 serving as a donor can coassemble with the alpha-helical M2 but not with M1, and M1 can coassemble with M2. No coassembly was observed between any of the segments and a membrane-embedded alpha-helical control peptide, pardaxin. The results are discussed in terms of their relevance to the proposed topology of the ROMK1 channel, and to general aspects of molecular recognition between membrane-bound polypeptides.

Membrane-active peptides participate in many cellular processes, and therefore knowledge of their mode of interaction with phospholipids is essential for understanding their biological function. Here we present a new methodology based on electron spin-echo envelope modulation to probe, at a relatively high resolution, the location of membrane-bound lytic peptides and to study their effect on the water concentration profile of the membrane. As a first example, we determined the location of the N-terminus of two membrane-active amphipathic peptides, the 26-mer bee venom melittin and a de novo designed 15-mer D,L-amino acid amphipathic peptide (5D-L9K6C), both of which are antimicrobial and bind and act similarly on negatively charged membranes. A nitroxide spin label was introduced to the N-terminus of the peptides and measurements were performed either in H2O solutions with deuterated model membranes or in D2O solutions with nondeuterated model membranes. The lipids used were dipalmitoyl phosphatidylcholine (DPPC) and phosphatidylglycerol (PG), (DPPC/PG (7:3w/w)), egg phosphatidylcholine (PC) and PG (PC/PG (7:3w/w)), and phosphatidylethanolamine (PE) and PG (PE/PG, 7:3w/w). The modulation induced by the 2H nuclei was determined and compared with a series of controls that produced a reference “ruler”. Actual estimated distances were obtained from a quantitative analysis of the modulation depth based on a simple model of an electron spin situated at a certain distance from the bottom of a layer with homogeneously distributed deuterium nuclei. The N-terminus of both peptides was found to be in the solvent layer in both the DPPC/PG and PC/PG membranes. For PE/PG, a further displacement into the solvent was observed. The addition of the peptides was found to change the water distribution in the membrane, making it “flatter” and increasing the penetration depth into the hydrophobic region.

Antimicrobial peptides are an essential part of the innate immune system of most living things and the understanding of the biophysical properties and the different mechanisms of action are crucial for the de-novo development of simple and effective analogs. More specifically, antimicrobial lipopeptides have been gaining increased attention because of the pressure for new antimicrobial agents against resistant pathogens. The addition of a lipophilic fatty acid has proven to be an effective method to increase the association of a peptide with the membrane, thus increasing the biological activity of certain peptide sequences. Previously, we reported that linear ultrashort cationic lipopeptides even as short as 4 amino acids have potent antimicrobial and antifungal properties. We described the minimum peptide length, and fatty acid length necessary for activity. Also, innate-immunity-like peptides were described that contained multiple histidine residues. Although these peptides consisted of 12 to 15 amino acids, these were less toxic to the host, and were lytic to numerous pathogens and cancer cells at slightly acidic environments. Here we report the design of an ultrashort histidine containing peptide whose antifungal activity could be significantly increased in a covalent trimeric form. Low micromolar activity was observed for Aspergillus fumigatus and Cryptococcus neoformans but not Candida albecans. Using transmission electron microscopy, we observed that this trimeric ultrashort histidine containing peptide formed distinct and differing nanostructures at pH 5 and 7, which could explain the activity differences. Since various organs or areas of the human body have a slightly acidic pH environment such as tumors, gastric lumen and lung-lining fluids in cystic fibrosis and asthma, understanding the importance of nanostructure-activity relationships of these pH dependent ultrashort peptides could lead to improvements in the delivery and administration of the peptides.

The bacterial outer membrane, lipopolysaccharide (LPS), can serve as a barrier that protects bacteria from antimicrobial peptides (AMPs). However, LPS can also activate immune cells that in sever cases may cause death. This activity can also be neutralized by several AMPs. However, it is difficult to determine common denominators required for antimicrobial and LPS neutralizing activities. To this end, we synthesized and investigated a series of 12-mer D,L-amino acid peptides and their fatty acid-conjugated analogs composed of Leu and Lys with increasing number of positive charges and decreasing hydrophobicities, and with preserved positions for the D-amino acids. The overall altered helical structure in the membrane is similar for all of them as determined by FTIR spectroscopy. All the peptides were tested for their antibacterial and hemolytic activity, their ability to permeate LPS vesicles, to neutralize LPS activation of macrophages, as well as their effect on LPS morphology, determined by negative staining electron microscopy. The data reveal that whereas antimicrobial activity increases linearly with the increase in the peptides' hydrophobicity, peptides with different hydrophobicities are endowed with similar LPS neutralizing activities. Besides its importance to the understanding of antimicrobial and LPS neutralizing activities, this study suggests the use of such diastereomers as potential templates for the development of simple molecules that carry out both types of functions.