The polar amino acid in the TatA transmembrane helix is not strictly necessary for protein function.

The twin-arginine translocation (Tat) pathway utilizes the proton-motive force to transport folded proteins across cytoplasmic membranes in bacteria and archaea, as well as across the thylakoid membrane in plants and the inner membrane in mitochondria. In most species, the minimal components required for Tat activity consist of three subunits, TatA, TatB, and TatC. Previous studies have shown that a polar amino acid is present at the N terminus of the TatA transmembrane helix (TMH) across many different species. In order to systematically assess the functional importance of this polar amino acid in the TatA TMH in Escherichia coli, we examined a complete set of 19-amino-acid substitutions. Unexpectedly, although the polar amino acid is preferred overall, our experiments suggest that it is not necessary for a functional TatA. Hydrophilicity and helix-stabilizing properties of this polar amino acid were found to be highly correlated with the Tat activity. Specifically, change in charge status of the amino acid side chain due to pH resulted in a shift in hydrophilicity, which was demonstrated to impact the Tat transport activity. Furthermore, we identified a four-residue motif at the N terminus of the TatA TMH by sequence alignment. Using a biochemical approach, we found that the N-terminal motif was functionally significant, with evidence indicating a potential role in the preference for utilizing different proton-motive force components. Taken together, these findings yield new insights into the functionality of TatA and its potential role in the Tat transport mechanism.

Electrochromic shift supports the membrane destabilization model of Tat-mediated transport and shows ion leakage during Sec transport.

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between "closed" and "opened" states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.

Hydrophobic mismatch is a key factor in protein transport across lipid bilayer membranes via the Tat pathway.

The twin-arginine translocation (Tat) pathway transports folded proteins across membranes in bacteria, thylakoids, plant mitochondria, and archaea. In most species, the active Tat machinery consists of three independent subunits: TatA, TatB, and TatC. TatA and TatB possess short transmembrane alpha helices (TMHs), both of which are only 15 residues long in Escherichia coli. Such short TMHs cause a hydrophobic mismatch between Tat subunits and the membrane bilayer, although the functional significance of this mismatch is unclear. Here, we sought to address the functional importance of the hydrophobic mismatch in the Tat transport mechanism in E. coli. We conducted three different assays to evaluate the effect of TMH length mutants on Tat activity and observed that the TMHs of TatA and TatB appear to be evolutionarily tuned to 15 amino acids, with activity dropping off following any modification of this length. Surprisingly, TatA and TatB with as few as 11 residues in their TMHs can still insert into the membrane bilayer, albeit with a decline in membrane integrity. These findings support a model of Tat transport utilizing localized toroidal pores that form when the membrane bilayer is thinned to a critical threshold. In this context, we conclude that the 15-residue length of the TatA and TatB TMHs can be seen as a compromise between the need for some hydrophobic mismatch to allow the membrane to reversibly reach the threshold thinness required for toroidal pore formation and the permanently destabilizing effect of placing even shorter helices into these energy-transducing membranes.

Membrane Permeabilization Mechanisms.

Many antimicrobial peptides are considered to kill microbes by permeabilizing cell membranes. This chapter summarizes the driving force of peptide binding to membranes; various mechanisms of lipid bilayer permeabilization including the barrel-stave, toroidal pore, and carpet models; and modes of permeabilization of bacterial and mammalian membranes.

The Mechanisms of Action of Cationic Antimicrobial Peptides Refined by Novel Concepts from Biophysical Investigations.

Even 30 years after the discovery of magainins, biophysical and structural investigations on how these peptides interact with membranes can still bear surprises and add new interesting detail to how these peptides exert their antimicrobial action. Early on, using oriented solid-state NMR spectroscopy, it was found that the amphipathic helices formed by magainins are active when being oriented parallel to the membrane surface. More recent investigations indicate that this in-planar alignment is also found when PGLa and magainin in combination exert synergistic pore-forming activities, where studies on the mechanism of synergistic interaction are ongoing. In a related manner, the investigation of dimeric antimicrobial peptide sequences has become an interesting topic of research which bears promise to refine our views how antimicrobial action occurs. The molecular shape concept has been introduced to explain the effects of lipids and peptides on membrane morphology, locally and globally, and in particular of cationic amphipathic helices that partition into the membrane interface. This concept has been extended in this review to include more recent ideas on soft membranes that can adapt to external stimuli including membrane-disruptive molecules. In this manner, the lipids can change their shape in the presence of low peptide concentrations, thereby maintaining the bilayer properties. At higher peptide concentrations, phase transitions occur which lead to the formation of pores and membrane lytic processes. In the context of the molecular shape concept, the properties of lipopeptides, including surfactins, are shortly presented, and comparisons with the hydrophobic alamethicin sequence are made.

Membrane pore formation at protein-lipid interfaces.

Pore-forming proteins (PFPs) interact with lipid bilayers to compromise membrane integrity. Many PFPs function by inserting a ring of oligomerized subunits into the bilayer to form a protein-lined hydrophilic channel. However, mounting evidence suggests that PFPs can also generate 'proteolipidic' pores by contributing to the fusion of inner and outer bilayer leaflets to form a toroidal structure. We discuss here toroidal pore formation by peptides including melittin, protegrin, and Alzheimer's Aß1-41, as well as by PFPs from several evolutionarily unrelated families: the colicin/Bcl-2 grouping including the pro-apoptotic protein Bax, actinoporins derived from sea anemones, and the membrane attack complex-perforin/cholesterol dependent cytolysin (MACPF/CDC) set of proteins. We also explore how the structure and biological role of toroidal pores might be investigated further.

Erlang flow of hydrophilic pore formation and closure events in a lipid bilayer during phase transition resulting from diffusion in the radius space.

The Smoluchowski equation with an energy profile of a special type and an assumed hydrophobic ("half") pore source term is used to describe the process of hydrophilic pore formation in a lipid bilayer at the gel-liquid phase transition. The source term reflects the occurrence of molecule packing defects in a lipid bilayer at phase transition. The time sequences of the pore formation and closure events are treated as non-stationary, second-order Erlang flows whose characteristics depend on the equation solution. The computed distributions of the time intervals between hydrophilic pores, and pore lifetimes agree with the previously published experimental interpulse interval and pulse duration histograms for the current fluctuations through planar bilayer membranes of DPPC immersed in a LiCl aqueous solution containing polyethylene glycol. Thus, the statistical analysis of pore formation and closure times leads us to conclude that firstly, the increased permeability of a lipid bilayer during the gel-liquid phase transition is accounted for by the emergence of additional hydrophobic defects in the heterogeneous structure of the bilayer and secondly, that the non-exponential distributions of the lipid channel closed and open times observed in experiments are evidence that the process of hydrophilic pore formation is not a one-step process but involves at least two dependent events.

Marine antimicrobial peptide arenicin adopts a monomeric twisted ß-hairpin structure and forms low conductivity pores in zwitterionic lipid bilayers.

Arenicins are 21-residue ß-hairpin antimicrobial peptides (AMPs) isolated from the marine lugworm Arenicola marina [Ovchinnikova et al., FEBS Lett. 2004;577:209-214]. The peptides have a high positive charge (+6) and display a broad spectrum of antimicrobial activities against bacteria and fungi. Arenicins adopt the monomeric highly twisted ß-hairpin in water or planar ß-structural dimers in anionic liposomes and detergent micelles. Until now, the interaction of cationic ß-structural AMPs with zwitterionic phospholipid bilayers mimicking eukaryotic membranes is not well understood. To study the structural basis of arenicins activity against eukaryotic cells, we investigated arenicin-2 in the solvents of low polarity (ethanol, 4% dioxane) and in zwitterionic soybean PC and PC/PE liposomes by CD and FTIR spectroscopy. It was shown that arenicin-2 adopted the twisted ß-hairpin structure in all the environments studied. Measurements of the Trp fluorescence and H→D exchange in soybean PC liposomes and boundary potential in the planar DPhPC bilayers confirmed the partitioning of the arenicin-2 monomers into interfacial region of the zwitterionic membranes. The low-conductivity (0.12 nS) arenicin-2 pores were detected in the DPhPC bilayers. The lifetime of the open state (up to 260 ms) was significantly longer than lifetime of low-conductivity (0.23 nS) pores previously described in partially anionic membranes (44 ms). The formation of narrow arenicin-2 pores without disruption of the membrane was discussed in the light of the disordered toroidal pore model previously proposed for ß-structural AMPs [Jean - Francois et al. Biophys. J. 2008;95:5748 - 5756]. A novel non-lytic mechanism of the arenicin-2 action was proposed.

Measuring kinetic drivers of pneumolysin pore structure.

Most membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins are thought to form pores in target membranes by assembling into pre-pore oligomers before undergoing a pre-pore to pore transition. Assembly during pore formation is into both full rings of subunits and incomplete rings (arcs). The balance between arcs and full rings is determined by a mechanism dependent on protein concentration in which arc pores arise due to kinetic trapping of the pre-pore forms by the depletion of free protein subunits during oligomerization. Here we describe the use of a kinetic assay to study pore formation in red blood cells by the MACPF/CDC pneumolysin from Streptococcus pneumoniae. We show that cell lysis displays two kinds of dependence on protein concentration. At lower concentrations, it is dependent on the pre-pore to pore transition of arc oligomers, which we show to be a cooperative process. At higher concentrations, it is dependent on the amount of pneumolysin bound to the membrane and reflects the affinity of the protein for its receptor, cholesterol. A lag occurs before cell lysis begins; this is dependent on oligomerization of pneumolysin. Kinetic dissection of cell lysis by pneumolysin demonstrates the capacity of MACPF/CDCs to generate pore-forming oligomeric structures of variable size with, most likely, different functional roles in biology.

Process of inducing pores in membranes by melittin.

Melittin is a prototype of the ubiquitous antimicrobial peptides that induce pores in membranes. It is commonly used as a molecular device for membrane permeabilization. Even at concentrations in the nanomolar range, melittin can induce transient pores that allow transmembrane conduction of atomic ions but not leakage of glucose or larger molecules. At micromolar concentrations, melittin induces stable pores allowing transmembrane leakage of molecules up to tens of kilodaltons, corresponding to its antimicrobial activities. Despite extensive studies, aspects of the molecular mechanism for pore formation remain unclear. To clarify the mechanism, one must know the states of the melittin-bound membrane before and after the process. By correlating experiments using giant unilamellar vesicles with those of peptide-lipid multilayers, we found that melittin bound on the vesicle translocated and redistributed to both sides of the membrane before the formation of stable pores. Furthermore, stable pores are formed only above a critical peptide-to-lipid ratio. The initial states for transient and stable pores are different, which implies different mechanisms at low and high peptide concentrations. To determine the lipidic structure of the pore, the pores in peptide-lipid multilayers were induced to form a lattice and examined by anomalous X-ray diffraction. The electron density distribution of lipid labels shows that the pore is formed by merging of two interfaces through a hole. The molecular property of melittin is such that it adsorbs strongly to the bilayer interface. Pore formation can be viewed as the bilayer adopting a lipid configuration to accommodate its excessive interfacial area.

Lipid-dependent pore formation by antimicrobial peptides arenicin-2 and melittin demonstrated by their proton transfer activity.

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, ß-structural arenicin-2 and α-helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light-induced electrochemical proton gradient ∆ΔpH. Addition of several nanomoles of the peptides lowers ∆ΔpH that is proximately indicative of the pore formation. The quantitative analysis of sigmoidal dependences of ∆pH on the peptides concentration was carried out using liposomes prepared from PC, PC/PE, PC/PE/PI and PC/PG. Substitution of PC-containing liposomes with PE-containing ones, having negative spontaneous curvature, reduced the PTA of α-helical melittin and increased that of ß-structural arenicin-2. This result indicates an essential difference in the pore formation by these peptides. Further increase of PTA in response to arenicin-2 (in contrast to melittin) was observed in the liposomes prepared from PC/PE/PI. The data analysis leads to the conclusion that PTA is influenced by (i) efficiency of the pore assemblage, which depends on the structure of pore-forming peptides, and the spontaneous curvature of lipids and (ii) the presence of mobile protons in the polar head groups of phospholipids.

The SMART model: Soft Membranes Adapt and Respond, also Transiently, in the presence of antimicrobial peptides.

Biophysical and structural studies of peptide-lipid interactions, peptide topology and dynamics have changed our view on how antimicrobial peptides insert and interact with membranes. Clearly, both the peptides and the lipids are highly dynamic, change and mutually adapt their conformation, membrane penetration and detailed morphology on a local and a global level. As a consequence, the peptides and lipids can form a wide variety of supramolecular assemblies in which the more hydrophobic sequences preferentially, but not exclusively, adopt transmembrane alignments and have the potential to form oligomeric structures similar to those suggested by the transmembrane helical bundle model. In contrast, charged amphipathic sequences tend to stay intercalated at the membrane interface where they cause pronounced disruptions of the phospholipid fatty acyl packing. At increasing local or global concentrations, the peptides result in transient membrane openings, rupture and ultimately lysis. Depending on peptide-to-lipid ratio, lipid composition and environmental factors (temperature, buffer composition, ionic strength, etc.), the same peptide sequence can result in a variety of those responses. Therefore, the SMART model has been introduced to cover the full range of possibilities. With such a view in mind, novel antimicrobial compounds have been designed from amphipathic polymers, peptide mimetics, combinations of ultra-short polypeptides with hydrophobic anchors or small designer molecules.

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains.

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

Mechanistic Insight into the Early Stages of Toroidal Pore Formation by the Antimicrobial Peptide Smp24.

The antimicrobial peptide Smp24, originally derived from the venom of Scorpio maurus palmatus, is a promising candidate for further drug development. However, before doing so, greater insight into the mechanism of action is needed to construct a reliable structure-activity relationship. The aim of this study was to specifically investigate the critical early stages of peptide-induced membrane disruption. Single-channel current traces were obtained via planar patch-clamp electrophysiology, with multiple types of pore-forming events observed, unlike those expected from the traditional, more rigid mechanistic models. To better understand the molecular-level structures of the peptide-pore assemblies underlying these observed conductance events, molecular dynamics simulations were used to investigate the peptide structure and orientation both before and during pore formation. The transition of the peptides to transmembrane-like states within disordered toroidal pores occurred due to a peptide-induced bilayer-leaflet asymmetry, explaining why pore stabilization does not always follow pore nucleation in the experimental observations. To fully grasp the structure-activity relationship of antimicrobial peptides, a more nuanced view of the complex and dynamic mechanistic behaviour must be adopted.

A Novel Cryptic Clostridial Peptide That Kills Bacteria by a Cell Membrane Permeabilization Mechanism.

This work reports detailed characteristics of the antimicrobial peptide Intestinalin (P30), which is derived from the LysC enzyme of Clostridium intestinale strain URNW. The peptide shows a broader antibacterial spectrum than the parental enzyme, showing potent antimicrobial activity against clinical strains of Gram-positive staphylococci and Gram-negative pathogens and causing between 3.04 ± 0.12 log kill for Pseudomonas aeruginosa PAO1 and 7.10 ± 0.05 log kill for multidrug-resistant Acinetobacter baumannii KPD 581 at a 5 µM concentration. Moreover, Intestinalin (P30) prevents biofilm formation and destroys 24-h and 72-h biofilms formed by Acinetobacter baumannii CRAB KPD 205 (reduction levels of 4.28 and 2.62 log CFU/mL, respectively). The activity of Intestinalin is combined with both no cytotoxicity and little hemolytic effect against mammalian cells. The nuclear magnetic resonance and molecular dynamics (MD) data show a high tendency of Intestinalin to interact with the bacterial phospholipid cell membrane. Although positively charged, Intestinalin resides in the membrane and aggregates into small oligomers. Negatively charged phospholipids stabilize peptide oligomers to form water- and ion-permeable pores, disrupting the integrity of bacterial cell membranes. Experimental data showed that Intestinalin interacts with negatively charged lipoteichoic acid (logK based on isothermal titration calorimetry, 7.45 ± 0.44), causes membrane depolarization, and affects membrane integrity by forming large pores, all of which result in loss of bacterial viability. IMPORTANCE Antibiotic resistance is rising rapidly among pathogenic bacteria, becoming a global public health problem that threatens the effectiveness of therapies for many infectious diseases. In this respect, antimicrobial peptides appear to be an interesting alternative to combat bacterial pathogens. Here, we report the characteristics of an antimicrobial peptide (of 30 amino acids) derived from the clostridial LysC enzyme. The peptide showed killing activity against clinical strains of Gram-positive and Gram-negative pathogens. Experimental data and computational modeling showed that this peptide forms transmembrane pores, directly engaging the negatively charged phospholipids of the bacterial cell membrane. Consequently, dissipation of the electrochemical gradient across cell membranes affects many vital processes, such as ATP synthesis, motility, and transport of nutrients. This kind of dysfunction leads to the loss of bacterial viability. Our firm conviction is that the presented study will be a helpful resource in searching for novel antimicrobial peptides that could have the potential to replace conventional antibiotics.

Lipid Microenvironment Modulates the Pore-Forming Ability of Polymyxin B.

The ability of polymyxin B, an antibiotic used to treat infections caused by multidrug-resistant Gram-negative bacteria as a last-line therapeutic option, to form ion pores in model membranes composed of various phospholipids and lipopolysaccharides was studied. Our data demonstrate that polymyxin B predominantly interacts with negatively charged lipids. Susceptibility decreases as follows: Kdo2-Lipid A >> DOPG ≈ DOPS >> DPhPG ≈ TOCL ≈ Lipid A. The dimer and hexamer of polymyxin B are involved in the pore formation in DOPG(DOPS)- and Kdo2-Lipid A-enriched bilayers, respectively. The pore-forming ability of polymyxin B significantly depends on the shape of membrane lipids, which indicates that the antibiotic produces toroidal lipopeptide-lipid pores. Small amphiphilic molecules diminishing the membrane dipole potential and inducing positive curvature stress were shown to be agonists of pore formation by polymyxin B and might be used to develop innovative lipopeptide-based formulations.

The effects of SDS at subsolubilizing concentrations on the planar lipid bilayer permeability: Two kinds of current fluctuations.

Detergent effects on lipid bilayers of artificial and biological membranes at subsolubilizing concentrations are known to include the membrane permeabilization which manifests itself through both a flip-flop of detergent molecules from the outer monolayer to the inner monolayer and the membrane leakage of entrapped solutes. We have explored the current fluctuations occurring in planar BLM of asolectin in the presence of ionic detergent SDS at subsolubilizing concentration. Two groups of current fluctuations which the average duration differs by two orders of magnitude can be distinguished. We assume that these differences in the duration of current fluctuations are associated with a different number of SDS molecules in the walls of the putative toroidal hydrophilic pores. We associated short pulses with the formation of short-lived lipid hydrophilic pores. Impulses of greater duration (steps) were associated with the formation of hydrophilic pores, the walls of which contain detergent. Taking into account the characteristics of these pores, we estimated the pore energy, as well as the edge energy of these two kinds of pores. We believe that the flip-flop of SDS molecules in liposomes is provided by long-lived pores, and the contents of the liposome leakage occurs through all pores.

Biophysical Investigations Elucidating the Mechanisms of Action of Antimicrobial Peptides and Their Synergism.

Biophysical and structural investigations are presented with a focus on the membrane lipid interactions of cationic linear antibiotic peptides such as magainin, PGLa, LL37, and melittin. Observations made with these peptides are distinct as seen from data obtained with the hydrophobic peptide alamethicin. The cationic amphipathic peptides predominantly adopt membrane alignments parallel to the bilayer surface; thus the distribution of polar and non-polar side chains of the amphipathic helices mirror the environmental changes at the membrane interface. Such a membrane partitioning of an amphipathic helix has been shown to cause considerable disruptions in the lipid packing arrangements, transient openings at low peptide concentration, and membrane disintegration at higher peptide-to-lipid ratios. The manifold supramolecular arrangements adopted by lipids and peptides are represented by the 'soft membranes adapt and respond, also transiently' (SMART) model. Whereas molecular dynamics simulations provide atomistic views on lipid membranes in the presence of antimicrobial peptides, the biophysical investigations reveal interesting details on a molecular and supramolecular level, and recent microscopic imaging experiments delineate interesting sequences of events when bacterial cells are exposed to such peptides. Finally, biophysical studies that aim to reveal the mechanisms of synergistic interactions of magainin 2 and PGLa are presented, including unpublished isothermal titration calorimetry (ITC), circular dichroism (CD) and dynamic light scattering (DLS) measurements that suggest that the peptides are involved in liposome agglutination by mediating intermembrane interactions. A number of structural events are presented in schematic models that relate to the antimicrobial and synergistic mechanism of amphipathic peptides when they are aligned parallel to the membrane surface.

Incomplete pneumolysin oligomers form membrane pores.

Pneumolysin is a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming proteins that are produced as water-soluble monomers or dimers, bind to target membranes and oligomerize into large ring-shaped assemblies comprising approximately 40 subunits and approximately 30 nm across. This pre-pore assembly then refolds to punch a large hole in the lipid bilayer. However, in addition to forming large pores, pneumolysin and other CDCs form smaller lesions characterized by low electrical conductance. Owing to the observation of arc-like (rather than full-ring) oligomers by electron microscopy, it has been hypothesized that smaller oligomers explain smaller functional pores. To investigate whether this is the case, we performed cryo-electron tomography of pneumolysin oligomers on model lipid membranes. We then used sub-tomogram classification and averaging to determine representative membrane-bound low-resolution structures and identified pre-pores versus pores by the presence of membrane within the oligomeric curve. We found pre-pore and pore forms of both complete (ring) and incomplete (arc) oligomers and conclude that arc-shaped oligomeric assemblies of pneumolysin can form pores. As the CDCs are evolutionarily related to the membrane attack complex/perforin family of proteins, which also form variably sized pores, our findings are of relevance to that class of proteins as well.