Dye-release assay for investigation of antimicrobial peptide activity in a competitive lipid environment

A dye-release method for investigating the effect of a competitive lipid environment on the activity of two membrane-disrupting antimicrobial peptides (AMP), maculatin 1.1 and aurein 1.2, is presented. The results support the general conclusion that AMP have greater affinity for negatively charged membranes, for example bacterial membranes, than for the neutral membrane surface found in eukaryotic cells, but only within a competitive lipid environment. Indeed, in a single-model membrane environment, both peptides were more potent against neutral vesicles than against charged vesicles. The approach was also used to investigate the effect of pre-incubating the peptides in a neutral lipid environment then introducing charged lipid vesicles. Maculatin was shown to migrate from the neutral lipid bilayers, where pores had already formed, to the charged membrane bilayers. This result was also observed for charged-to-charged bilayers but, interestingly, not for neutral-to-neutral lipid interfaces. Aurein was able to migrate from either lipid environment, indicating weaker binding to lipid membranes, and a different molecular mechanism for lysis of lipid bilayers. Competitive lipid environments could be used to assess other critical conditions that modulate the activity of membrane peptides or proteins.     

Membrane insertion and lateral mobility of synthetic amphiphilic signal peptides in lipid model membranes

Amphiphilic signal sequences with the potential to form alpha-helices with a polar, charged face and an apolar face are common in proteins which are imported into mitochondria, in the PTS permeases of bacteria, and in bacterial rhodopsins. Synthetic peptides of such sequences partition into the surface region of lipid membranes where they can adopt different secondary structures. A finely controlled balance of electrostatic and hydrophobic interactions determines the 'affinity' of amphiphilic signal peptides for lipid membranes, as well as the structure, orientation and depth of penetration of these peptides in lipid bilayer membranes. The ability of an individual peptide to associate with lipid bilayer membranes in several different modes is, most likely, a general feature of amphiphilic signal peptides and is reflected in several common physical properties of their amino acid sequences.     

Binding of peptides corresponding to the carboxy-terminal region of human-β-defensins-1-3 with model membranes investigated by isothermal titration calorimetry

Human-β-defensins HBD-1-3 are important components of the innate immune system. Synthetic peptides Phd-1-3 with a single disulphide bond, spanning the cationic C-terminal region of HBD-1-3, have antimicrobial activity. The interaction of Phd-1-3 with model membranes was investigated using isothermal titration calorimetry (ITC) and steady-state fluorescence polarization to understand the biophysical basis for the mechanism of antimicrobial action. Calorimetric titration of POPE:POPG (7:3) vesicles with peptides at 25°C and 37°C showed complex profiles with two distinct regions of heat changes. The data indicate binding of Phd-1-3 at 37°C to both negative and zwitterionic lipid vesicles is exothermic with low enthalpy values (ΔH~-1.3 to -2.8kcal/mol) as compared to amphipathic helical antibacterial peptides. The adsorption of peptides to negatively charged lipid membranes is modulated by electrostatic interactions that are described by surface partition equilibrium model using Gouy-Chapman theory. However, this model could not explain the isotherms of peptide binding to zwitterionic lipid vesicles. Fluorescence polarization of TMA-DPH (1-[4-(trimethylammonio) phenyl]-6-phenyl-1,3,5-hexatriene) and DPH (1,6-diphenyl-1,3,5-hexatriene) located in the head group and acyl chain region respectively, indicates that the peptides interact with interfacial region of negatively charged membranes. Based on the results obtained, we conclude that adsorption of cationic peptides Phd-1-3 on lipid surface do not result in conformational change or pore formation. It is proposed that interaction of Phd-1-3 with the negatively charged lipid head group causes membrane destabilization, which in turn affects the efficient functioning of cytoplasmic membrane proteins in bacteria, resulting in cell death.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Alzheimer Aβ peptide interactions with lipid membranes

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Membrane association and selectivity of the antimicrobial peptide NK‐2: a molecular dynamics simulation study

In an effort to better understand the initial mechanism of selectivity and membrane association of the synthetic antimicrobial peptide NK‐2, we have applied molecular dynamics simulation techniques to elucidate the interaction of the peptide with the membrane interfaces. A homogeneous dipalmitoylphosphatidylglycerol (DPPG) and a homogeneous dipalmitoylphosphatidylethanolamine (DPPE) bilayers were taken as model systems for the cytoplasmic bacterial and human erythrocyte membranes, respectively. The results of our simulations on DPPG and DPPE model membranes in the gel phase show that the binding of the peptide, which is considerably stronger for the negatively charged DPPG lipid bilayer than for the zwitterionic DPPE, is mostly governed by electrostatic interactions between negatively charged residues in the membrane and positively charged residues in the peptide. In addition, a characteristic distribution of positively charged residues along the helix facilitates a peptide orientation parallel to the membrane interface. Once the peptides reside close to the membrane surface of DPPG with the more hydrophobic side chains embedded into the membrane interface, the peptide initially disturbs the respective bilayer integrity by a decrease of the order parameter of lipid acyl chain close to the head group region, and by a slightly decrease in bilayer thickness. We found that the peptide retains a high content of helical structure on the zwitterionic membrane‐water interface, while the loss of α‐helicity is observed within a peptide adsorbed onto negatively charged lipid membranes. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of synthetic peptides corresponding to the scaffolding domain of Caveolin-3 with model membranes

Caveolin-1 and -3 are among the few proteins in which the functional domains are contiguous and modular. The interaction of synthetic peptides spanning the scaffolding domain of caveolin-3 with model membranes has been investigated. The peptides include the scaffolding domain, the aromatic and positively charged residues at the C-terminal end of this domain as well as deletion of three amino acids TFT, observed in certain patients with limb girdle muscular dystrophy. All of the peptides appear to be peripherally bound to the bilayer surface. However, no preferential binding to sphingomyelin and cholesterol-containing lipid vesicles was observed. Deletion of TFT appears to affect the association with lipid vesicles compared with the native sequence. Association with lipids decreases considerably when TFT as well as the aromatic-rich segment YWFYR, which occurs at the extreme C-terminus of the scaffolding domain, are deleted.     

Spin-label electron spin resonance studies on the interactions of lysine peptides with phospholipid membranes.

The interactions of lysine oligopeptides with dimyristoyl phosphatidylglycerol (DMPG) bilayer membranes were studied using spin-labeled lipids and electron spin resonance spectroscopy. Tetralysine and pentalysine were chosen as models for the basic amino acid clusters found in a variety of cytoplasmic membrane-associating proteins, and polylysine was chosen as representative of highly basic peripherally bound proteins. A greater motional restriction of the lipid chains was found with increasing length of the peptide, while the saturation ratio of lipids per peptide was lower for the shorter peptides. In DMPG and dimyristoylphosphatidylserine host membranes, the perturbation of the lipid chain mobility by polylysine was greater for negatively charged spin-labeled lipids than for zwitterionic lipids, but for the shorter lysine peptides these differences were smaller. In mixed bilayers composed of DMPG and dimyristoylphosphatidylcholine, little difference was found in selectivity between spin-labeled phospholipid species on binding pentalysine. Surface binding of the basic lysine peptides strongly reduced the interfacial pK of spin-labeled fatty acid incorporated into the DMPG bilayers, to a greater extent for polylysine than for tetralysine or pentalysine at saturation. The results are consistent with a predominantly electrostatic interaction with the shorter lysine peptides, but with a closer surface association with the longer polylysine peptide.     

Alzheimer Aβ peptide interactions with lipid membranes: Fibrils,oligomers and polymorphic amyloid channels

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Minimal Effect of Lipid Charge on Membrane Miscibility Phase Behavior in Three Ternary Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Domain formation induced by the adsorption of charged proteins on mixed lipid membranes

Peripheral proteins can trigger the formation of domains in mixed fluid-like lipid membranes. We analyze the mechanism underlying this process for proteins that bind electrostatically onto a flat two-component membrane, composed of charged and neutral lipid species. Of particular interest are membranes in which the hydrocarbon lipid tails tend to segregate owing to nonideal chain mixing, but the (protein-free) lipid membrane is nevertheless stable due to the electrostatic repulsion between the charged lipid headgroups. The adsorption of charged, say basic, proteins onto a membrane containing anionic lipids induces local lipid demixing, whereby charged lipids migrate toward (or away from) the adsorption site, so as to minimize the electrostatic binding free energy. Apart from reducing lipid headgroup repulsion, this process creates a gradient in lipid composition around the adsorption zone, and hence a line energy whose magnitude depends on the protein's size and charge and the extent of lipid chain nonideality. Above a certain critical lipid nonideality, the line energy is large enough to induce domain formation, i.e., protein aggregation and, concomitantly, macroscopic lipid phase separation. We quantitatively analyze the thermodynamic stability of the dressed membrane based on nonlinear Poisson-Boltzmann theory, accounting for both the microscopic characteristics of the proteins and lipid composition modulations at and around the adsorption zone. Spinodal surfaces and critical points of the dressed membranes are calculated for several different model proteins of spherical and disk-like shapes. Among the models studied we find the most substantial protein-induced membrane destabilization for disk-like proteins whose charges are concentrated in the membrane-facing surface. If additional charges reside on the side faces of the proteins, direct protein-protein repulsion diminishes considerably the propensity for domain formation. Generally, a highly charged flat face of a macroion appears most efficient in inducing large compositional gradients, hence a large and unfavorable line energy and consequently lateral macroion aggregation and, concomitantly, macroscopic lipid phase separation.     

Interaction of mutant alkaline phosphatase precursors with membrane phospholipids in vivo and in vitro.

Positively charged amino acid residues in the N-terminal domain of the signal peptides of secreted proteins are thought to interact with negatively charged anionic phospholipids during the initiation of secretion. To test this hypothesis, substitutions of the uncharged Ala or the negatively charged Glu residue for the positively charged Lys-20 of the N-terminus of the signal peptide of Escherichia coli alkaline phosphatase were introduced using a modified method of oligonucleotide-directed mutagenesis. We found that Lys-20 is involved in the interaction of the signal peptide with anionic phospholipids in vivo and effects the efficiency of insertion of the signal peptide of isolated precursor into model phospholipid membranes in vitro. We also show that the efficiency of signal peptide insertion into the lipid bilayer depends on the fluidity of the bilayer.     

Analytic Approaches to Stochastic Gene Expression in Multicellular Systems

Giant unilamellar vesicles composed of a ternary mixture of phospholipids and cholesterol exhibit coexisting liquid phases over a range of temperatures and compositions. A significant fraction of lipids in biological membranes are charged. Here, we present phase diagrams of vesicles composed of phosphatidylcholine (PC) lipids, which are zwitterionic; phosphatidylglycerol (PG) lipids, which are anionic; and cholesterol (Chol). Specifically, we use DiPhyPG-DPPC-Chol and DiPhyPC-DPPG-Chol. We show that miscibility in membranes containing charged PG lipids occurs over similarly high temperatures and broad lipid compositions as in corresponding membranes containing only uncharged lipids, and that the presence of salt has a minimal effect. We verified our results in two ways. First, we used mass spectrometry to ensure that charged PC/PG/Chol vesicles formed by gentle hydration have the same composition as the lipid stocks from which they are made. Second, we repeated the experiments by substituting phosphatidylserine for PG as the charged lipid and observed similar phenomena. Our results consistently support the view that monovalent charged lipids have only a minimal effect on lipid miscibility phase behavior in our system.     

Resolving the kinetics of lipid, protein and peptide diffusion in membranes

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.     

Rhodopsin and other proteins in artificial lipid membranes.

Some basic aspects of incorporation of hydrophobic peptides and proteins in artificial lipid membranes are discussed. As examples valinomycin as a carrier model and gramicidin A as a channel former in lipid vesicles and in planar lipid membranes are presented. In the second part of the lecture some examples of incorporation of membrane proteins into lipid vesicles and planar lipid membranes are reported. The interaction with artificial lipid membranes of the Ca++ ATPase from the sarcoplasmic reticulum, of Rhodopsin, and of Bacteriorhodopsin is presented.     

Characterization of a putative fusogenic sequence in the E2 hepatitis G virus protein

With the aim of better understanding the fusion process mediated by the envelope proteins of the hepatitis G virus (HGV/GBV-C), we have investigated the interaction with model membranes of two overlapping peptides [(267-284) and (279-298)] belonging to the E2 structural protein. The peptides were compared for their ability to perturb lipid bilayers by means of different techniques such as differential scanning calorimetry and fluorescence spectroscopy. Furthermore, the conformational behaviour of the peptides in different membrane environments was studied by Fourier-transform infrared spectroscopy and circular dichroism. The results showed that only the E2(279-298) peptide sequence was able to bind with high affinity to negatively charged membranes, to permeabilize efficiently negative lipid bilayers, to induce haemolysis, and to promote inter-vesicle fusion. This fusogenic activity could be related to the induced peptide conformation upon interaction with the target membrane.     

Lipid demixing and protein-protein interactions in the adsorption of charged proteins on mixed membranes

The adsorption free energy of charged proteins on mixed membranes, containing varying amounts of (oppositely) charged lipids, is calculated based on a mean-field free energy expression that accounts explicitly for the ability of the lipids to demix locally, and for lateral interactions between the adsorbed proteins. Minimization of this free energy functional yields the familiar nonlinear Poisson-Boltzmann equation and the boundary condition at the membrane surface that allows for lipid charge rearrangement. These two self-consistent equations are solved simultaneously. The proteins are modeled as uniformly charged spheres and the (bare) membrane as an ideal two-dimensional binary mixture of charged and neutral lipids. Substantial variations in the lipid charge density profiles are found when highly charged proteins adsorb on weakly charged membranes; the lipids, at a certain demixing entropy penalty, adjust their concentration in the vicinity of the adsorbed protein to achieve optimal charge matching. Lateral repulsive interactions between the adsorbed proteins affect the lipid modulation profile and, at high densities, result in substantial lowering of the binding energy. Adsorption isotherms demonstrating the importance of lipid mobility and protein-protein interactions are calculated using an adsorption equation with a coverage-dependent binding constant. Typically, at bulk-surface equilibrium (i.e., when the membrane surface is "saturated" by adsorbed proteins), the membrane charges are "overcompensated" by the protein charges, because only about half of the protein charges (those on the hemispheres facing the membrane) are involved in charge neutralization. Finally, it is argued that the formation of lipid-protein domains may be enhanced by electrostatic adsorption of proteins, but its origin (e.g., elastic deformations associated with lipid demixing) is not purely electrostatic.     

Interaction of heat shock protein 70 with membranes depends on the lipid environment

Heat shock proteins (hsp) are well recognized for their protein folding activity. Additionally, hsp expression is enhanced during stress conditions to preserve cellular homeostasis. Hsp are also detected outside cells, released by an active mechanism independent of cell death. Extracellular hsp appear to act as signaling molecules as part of a systemic response to stress. Extracellular hsp do not contain a consensus signal for their secretion via the classical ER-Golgi compartment. Therefore, they are likely exported by an alternative mechanism requiring translocation across the plasma membrane. Since Hsp70, the major inducible hsp, has been detected on surface of stressed cells, we propose that membrane interaction is the first step in the export process. The question that emerges is how does this charged cytosolic protein interact with lipid membranes? Prior studies have shown that Hsp70 formed ion conductance pathways within artificial lipid bilayers. These early observations have been extended herewith using a liposome insertion assay. We showed that Hsp70 selectively interacted with negatively charged phospholipids, particularly phosphatidyl serine (PS), within liposomes, which was followed by insertion into the lipid bilayer, forming high-molecular weight oligomers. Hsp70 displayed a preference for less fluid lipid environments and the region embedded into the lipid membrane was mapped toward the C-terminus end of the molecule. The results from our studies provide evidence of an unexpected ability of a large, charged protein to become inserted into a lipid membrane. This observation provides a new paradigm for the interaction of proteins with lipid environments. In addition, it may explain the export mechanism of an increasing number of proteins that lack the consensus secretory signals.     

Location and dynamics of basic peptides at the membrane interface: electron paramagnetic resonance spectroscopy of tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid-labeled peptides

The attractive interaction between basic protein domains and membranes containing acidic lipids is critical to the membrane attachment of many proteins involved in cell signaling. In this study, a series of charged model peptides containing lysine, phenylalanine, and the spin-labeled amino acid tetramethyl-piperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) were synthesized, and electron paramagnetic resonance (EPR) spectroscopy was used to determine their position on the membrane interface and free energy of binding. When membrane-bound, peptides containing only lysine and TOAC assume an equilibrium position within the aqueous double layer at a distance of approximately 5 A from the membrane interface, a result that is consistent with recent computational work. Substitution of two or more lysine residues by phenylalanine dramatically slows the backbone diffusion of these peptides and shifts their equilibrium position by 13-15 A so that the backbone lies several angstroms below the level of the lipid phosphate. These results are consistent with the hypothesis that the position and free energy of basic peptides when bound to membranes are determined by a long-range Coulombic attraction, the hydrophobic effect, and a short-range desolvation force. The differences in binding free energy within this set of charged peptides is not well accounted for by the simple addition of free energies based upon accepted side chain partition free energies, a result that appears to be in part due to differences in membrane localization of these peptides.     

α-Helical transmembrane peptides: A “Divide and Conquer” approach to membrane proteins

α-Helical membrane proteins fulfill many vital roles in all living cells and constitute the majority of drug targets. However, their relevance is in no way paralleled by our current understanding of their structures and functions. This is because membrane proteins present a number of experimental obstacles that are difficult to surmount by classical methods developed for water-soluble proteins. Moreover, membrane proteins are not only challenging on their very own but, when embedded in a biological membrane, also reside in an outstandingly complex milieu. These difficulties have fostered a “divide and conquer” approach, in which a membrane protein is dissected into shorter and easier-to-handle transmembrane (TM) peptides. Under suitable conditions, such peptides fold independently and retain many of the properties displayed in the context of the full-length parent protein.This contribution reviews some of the most notable insights into α-helical membrane proteins gleaned from experiments on protein-derived TM peptides. We recapitulate some peculiar properties of lipid bilayers that render them such a complex and unique environment and discuss generic features pertaining to hydrophobic peptides derived from α-helical membrane proteins. The main part of the review is devoted to a critical discussion of particularly interesting examples of TM peptides studied in membrane-mimetic systems of increasing complexity: isotropic solvents, detergent micelles, lipid bilayers, and biological membranes. The unifying theme is to explore to what extent TM peptides in combination with different membrane-mimetic systems can aid in advancing our knowledge and comprehension of α-helical membrane proteins as well as in developing new pharmacological tools.