Mechanism Of Membrane Interaction Research Articles

Investigations on Membrane Perturbation by Chrysin and Its Copper Complex Using Self-Assembled Lipid Bilayers

The mechanism of membrane interactions of most of the flavonoids in the presence of transition-metal ions is not well-understood. To understand this phenomenon, the present work aims to synthesize a chrysin-copper complex at room temperature and investigate its influence on the electrical characteristics of planar lipid bilayers. The chrysin-copper complex was characterized by various spectroscopic techniques and was found to have a metal/ligand ratio of 1:2 and of cationic nature. Its ability to inhibit 1,1'-diphenyl-2-picrylhydrazyl (DPPH) radicals was not significant at alkaline pH because of the involvement of the 5-hydroxy group in coordination with the copper ion compared to its parent flavonoid, chrysin (p < 0.05). The addition of different concentrations (20-100 μM) of chrysin and the chrysin-copper complex to lipid bilayers decreases the resistance, indicating a strong surface interaction and partial insertion into the bilayer near the lipid-water interface. The dose-dependent reduction in resistance as a result of the chrysin-copper complex is more pronounced in comparison to chrysin, implying that the bulkier and charged chrysin-copper complex displays greater ability to distort the lipid bilayer architecture. These conclusions were further confirmed by curcumin-loaded liposome permeabilization studies, where both chrysin and its Cu(II) complex increased the fluidity in a dose-dependent manner. However, the extent of fluidization by the chrysin-copper complex was nearly twice that of chrysin alone (p < 0.05). The implications of these surface interactions of chrysin and its copper complex on cell membranes were studied using a hypotonic hemolysis assay. Our results demonstrate that, at low concentrations (20 μM), the chrysin-copper complex exhibited twice the protection against hypotonic stress-induced membrane disruption when compared to chrysin. However, this stabilizing effect gradually decreased and became comparable to chrysin at higher concentrations. This biphasic behavior of the chrysin-copper complex could further be explored for therapeutic applications.

Structure and Mechanism of the Saposin-like Domain of a Plant Aspartic Protease

Many plant aspartic proteases contain an additional sequence of ~100 amino acids termed the plant-specific insert, which is involved in host defense and vacuolar targeting. Similar to all saposin-like proteins, the plant-specific insert functions via protein-membrane interactions; however, the structural basis for such interactions has not been studied, and the nature of plant-specific insert-mediated membrane disruption has not been characterized. In the present study, the crystal structure of the saposin-like domain of potato aspartic protease was resolved at a resolution of 1.9 Å, revealing an open V-shaped configuration similar to the open structure of human saposin C. Notably, vesicle disruption activity followed Michaelis-Menten-like kinetics, a finding not previously reported for saposin-like proteins including plant-specific inserts. Circular dichroism data suggested that secondary structure was pH-dependent in a fashion similar to influenza A hemagglutinin fusion peptide. Membrane effects characterized by atomic force microscopy and light scattering indicated bilayer solubilization as well as fusogenic activity. Taken together, the present study is the first report to elucidate the membrane interaction mechanism of plant saposin-like domains whereby pH-dependent membrane interactions resulted in bilayer fusogenic activity that probably arose from a viral type pH-dependent helix-kink-helix motif at the plant-specific insert N terminus.

A new approach to detect and study ion channel formation in microBLMs

In this paper, an innovative and versatile approach to detect pore formation in microBLMs and to investigate their ion selectivity is described. For the first time, both electrochemical impedance spectroscopy and conductivity measurements are performed on the same membrane, providing a simple, long-lasting and cheap method which can find applications in various ion channel-involving research fields. We make use of this improved experimental procedure to obtain for the first time a range of values to characterize homogeneous polycarbonate-supported microBLMs. To test the validity of our method, the well-known, channel-forming peptide gramicidin D was first employed. Then, we applied our approach to the study of the still unclear membrane interaction mechanisms of the peptides trichogin GA IV and phospholamban, finding that both can form ion channels in biomimetic models. The phospholamban-generated ion channels could be a promising therapeutic target in heart failures and other cardiac diseases. Our methodology might also be useful for detecting new drugs with ion-channel blocking action.

Role of temperature-independent lipoplex–cell membrane interactions in the efficiency boost of multicomponent lipoplexes

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Influence of C-Terminal Amidation on the Efficacy of Modelin-5

To gain insight into the effects of amidation on the mechanism of membrane interaction, we studied two peptides modelin-5-COOH and modelin-5-CONH(2) and found they exhibit high surface activities (23.2 and 27.1 mN/m, respectively). When they were tested against Escherichia coli, amidation was seen to increase efficacy approximately 10-fold. Our results demonstrated that both peptides adopted low levels of α-helix in solution (<20%); however, in the presence of E. coli lipid extract, modelin-5-CONH(2) had a greater propensity (69%) than modelin-5-COOH (32%) to generate α-helical structure. The binding coefficient for both peptides was ∼10 μM, and the Hill coefficient approximated 1, suggesting that for both peptides the interactions with E. coli membranes were monomeric and comparable in strength. The peptides showed a clear preference for anionic lipid, with monolayer data showing that enhanced levels of helicity were associated with a greater pressure change (∼6 mN/m). Use of fluorescein-phosphatidylethanolamine showed the amidated version was able to generate greater levels of membrane disruption, which was confirmed by thermodynamic analysis. The data would imply that both peptides are able to initially bind to bilayer structures, but upon binding, the amidation stabilizes helix formation. This would be expected to help overcome a key rate-limiting step and generate higher local concentrations of peptide at the bilayer interface, which in turn would be predicted to increase efficacy.

Identification and structural characterization of FYVE domain-containing proteins of Arabidopsis thaliana

BackgroundFYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)P). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded β-sheets, and a C-terminal α-helix, which houses eight conserved Zn2+ ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated.ResultsWe carried out an extensive examination of all Arabidopsis FYVE domains, including their identification, classification, molecular modeling and biophysical characterization using computational approaches. Our classification of fifteen Arabidopsis FYVE proteins at the outset reveals unique domain architectures for FYVE containing proteins, which are not paralleled in other organisms. Detailed sequence analysis and biophysical characterization of the structural models are used to predict membrane interaction mechanisms previously described for other FYVE domains and their subtle variations as well as novel mechanisms that seem to be specific to plants.ConclusionsOur study contributes to the understanding of the molecular basis of FYVE-based membrane targeting in plants on a genomic scale. The results show that FYVE domain containing proteins in plants have evolved to incorporate significant differences from those in other organisms implying that they play a unique role in plant signaling pathways and/or play similar/parallel roles in signaling to other organisms but use different protein players/signaling mechanisms.

Trp2313-His2315 of Factor VIII C2 Domain Is Involved in Membrane Binding

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 A) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed beta-strand of the C2 domain, Trp(2313)-His(2315). This result indicates that the Trp(2313)-His(2315) segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

A Single Molecule Fluorescence Study on the Consequences of Alpha-Synuclein Oxidation

Parkinson's Disease (PD) is a neurodegenerative disorder characterized by the deposition of fibrillar amyloid inclusions in the substantia nigra region of the brain. Aggregation of alpha-synuclein (aS), an abundant presynaptic protein, is thought to play an essential role in the pathogenesis of PD. Oligomeric intermediates of the aggregation process have been implicated in neuronal cell death, possibly by compromising cell membrane integrity. Oxidative stress appears to be directly involved in the pathogenesis of PD; indeed, extensive accumulation of nitrated aS has been detected in amyloid aggregates in PD post-mortem brain tissue. In vitro, oxidative modifications to aS can inhibit fibrillation and lead to the build-up of stable oligomers, which may cause increased toxicity. Here, we use single molecule fluorescence techniques (fluorescence correlation spectroscopy and single molecule Förster energy transfer) to investigate the influence of oxidative modifications on the molecular mechanisms of aS aggregation and membrane interaction. aS is unstructured in solution but residues 1-90 form an alpha-helix upon binding lipid bilayers. Tyrosine nitration leads to decreased binding of aS to lipid vesicles, which might entail a loss of aS native function. Interestingly, we find that nitration of tyrosines located at the C-terminus of the protein, which stays unstructured upon membrane binding, can modulate the affinity of the N-terminus. Another consequence of nitrative insult to the protein is the formation of di- and oligomeric aS species by di-tyrosine cross-linking. We find that protein cross-linking does not perturb the protein's ability to form an alpha-helix upon membrane binding, although the binding affinity is altered. Nitrative stress has been implicated to be involved in PD pathology and the characterization of its effects on aS conformation and membrane interaction will help to refine our understanding of the toxic form(s) of aS.

Lipid reorganization induced by membrane-active peptides probed using differential scanning calorimetry

The overlapping biological behaviors between some cell penetrating peptides (CPPs) and antimicrobial peptides (AMPs) suggest both common and different membrane interaction mechanisms. We thus explore the capacity of selected CPPs and AMPs to reorganize the planar distribution of binary lipid mixtures by means of differential scanning calorimetry (DSC). Additionally, membrane integrity assays and circular dichroism (CD) experiments were performed. Two CPPs (Penetratin and RL16) and AMPs belonging to the dermaseptin superfamily (Drs B2 and C-terminal truncated analog [1–23]-Drs B2 and two plasticins DRP-PBN2 and DRP-PD36KF) were selected. Herein we probed the impact of headgroup charges and acyl chain composition (length and unsaturation) on the peptide/lipid interaction by using binary lipid mixtures. All peptides were shown to be α-helical in all the lipid mixtures investigated, except for the two CPPs and [1–23]-Drs B2 in the presence of zwitterionic lipid mixtures where they were rather unstructured. Depending on the lipid composition and peptide sequence, simple binding to the lipid surface that occur without affecting the lipid distribution is observed in particular in the case of AMPs. Recruitments and segregation of lipids were observed, essentially for CPPs, without a clear relationship between peptide conformation and their effect in the lipid lateral organization. Nonetheless, in most cases after initial electrostatic recognition between the peptide charged amino acids and the lipid headgroups, the lipids with the lowest phase transition temperature were selectively recruited by cationic peptides while those with the highest phase transition were segregated. Membrane activities of CPPs and AMPs could be thus related to their preferential interactions with membrane defects that correspond to areas with marked fluidity. Moreover, due to the distinct membrane composition of prokaryotes and eukaryotes, lateral heterogeneity may be differently affected by cationic peptides leading to either uptake or/and antimicrobial activities.

Comparison of the membrane interaction mechanism of two antimicrobial RNases: RNase 3/ECP and RNase 7

Eosinophil cationic protein (ECP/RNase 3) and the skin derived ribonuclease 7 (RNase 7) are members of the RNase A superfamily. RNase 3 is mainly expressed in eosinophils whereas RNase 7 is primarily secreted by keratinocytes. Both proteins present a broad-spectrum antimicrobial activity and their bactericidal mechanism is dependent on their membrane destabilizing capacities. Using phospholipid vesicles as membrane models, we have characterized the protein membrane association process. Confocal microscopy experiments using giant unilamellar vesicles illustrate the morphological changes of the liposome population. By labelling both lipid bilayers and proteins we have monitored the kinetic of the process. The differential protein ability to release the liposome aqueous content was evaluated together with the micellation and aggregation processes. A distinct morphology of the protein/lipid aggregates was visualized by transmission electron microscopy and the proteins overall secondary structure in a lipid microenvironment was assessed by FTIR. Interestingly, for both RNases the membrane interaction events take place in a different behaviour and timing: RNase 3 triggers first the vesicle aggregation, while RNase 7 induces leakage well before the aggregation step. Their distinct mechanism of action at the membrane level may reflect different in vivo antipathogen functions.

Effects of Oxidative Stress on Aggregation and Membrane Interaction of alpha-Synuclein Characterized by Single Molecule Fluorescence

Oxidative stress has been implicated as a major contributing factor to Parkinson's Disease (PD), a neurodegenerative disorder characterized by the deposition of fibrillar amyloid inclusions in the substantia nigra. The primary protein component of these inclusions is alpha-synuclein (aS), an abundant presynaptic protein, whose natural functions have not yet been resolved but presumably involve synaptic vesicle trafficking. Aggregation of amyloid proteins involves sampling of heterogeneous conformational and oligomeric intermediates, and it is actually these species that have been implicated to be responsible for neuronal cell death, possibly by compromising cell membrane integrity. Here, we use single molecule fluorescence techniques (fluorescence correlation spectroscopy and single molecule Forster energy transfer) to investigate the influence of oxidative modifications to both the protein and the lipid matrix on the molecular mechanisms of aS aggregation and membrane interaction. We find that oxidative modification to either protein or lipid leads to a decrease of aS vesicle binding, with the extent of decrease being dependent on the lipid matrix. As aS natural functions most likely involve synaptic vesicle binding, these results might indicate a loss of aS function due to oxidative stress. Further, oxidized aS shows a different aggregation behavior and does not form amyloid fibrils. The systematic characterization of the effects of oxidation on aS aggregation and membrane interaction will help to refine our understanding of the toxic form(s) of aS in order to identify cellular targets for the design of therapeutics to treat or prevent PD.

Membrane Localization of S4 Transmembrane Segment of Voltage-Gated Ion Channels

Voltage sensor domain (VSD) plays a key role in the channel conductance of the voltage-gated ion channels. Out of four transmembrane segments in VSD, cationic fourth transmembrane segment (S4) is the principal voltage sensor. Various models based on X-ray crystallography and site directed mutagenesis studies have shown that S4 segment, in response to the membrane potential, undergoes a conformational rotatory motion in the hydrophobic core of the lipid bilayer. Functional studies using whole-cell/ patch clamp techniques have detailed the conductance properties of the channel. However, the exact mechanism of membrane interaction and orientation of the cationic S4 segment in the non-polar lipid bilayer is yet to be understood. Direct experimental evidence using native S4 segment and liposomes, without any other energetic cofactors, would allow a direct test of the underlying mechanism of localization of S4 in the lipid bilayer. We used various fluorescence techniques to study the interaction and penetrative depth of the native S4 peptide in the lipid bilayer. Detail information concerning autonomous partition of S4 peptides in lipids will be discussed.

Mechanism of alpha-synuclein oligomerization and membrane interaction: theoretical approach to unstructured proteins studies

Misfolding and oligomerization of unstructured proteins is involved in the pathogenesis of Parkinson's disease (PD), Alzheimer's disease, Huntington's disease, and other neurodegenerative disorders. Elucidation of possible conformations of these proteins and their interactions with the membrane is necessary to understand the molecular mechanisms of neurodegeneration. We developed a strategy that makes it possible to elucidate the molecular mechanisms of α-synuclein aggregation—a key molecular event in the pathogenesis of PD. This strategy can be also useful for the study of other unstructured proteins involved in neurodegeneration. The results of these theoretical studies have been confirmed with biochemical and electrophysiological studies. Our studies provide insights into the molecular mechanism for PD initiation and progression, and provide a useful paradigm for identifying possible therapeutic interventions through computational modeling.

Dissecting the Membrane Binding and Insertion Kinetics of a pHLIP Peptide

When it is bound to lipid bilayers, the conformation and location of the membrane pH (low) insertion peptide (pHLIP) depend on pH. This unique feature allows us to explicitly measure the kinetics leading to different membrane-bound states of pHLIP using a model membrane and stopped-flow technique. Our results show that the membrane association kinetics of pHLIP are multiexponential and are consistent with a parallel membrane interaction mechanism. Interestingly, our results also show that the overall rate at which the membrane-inserted state is formed is almost identical to that of formation of the surface-bound state, while prebinding slows the rate of peptide insertion.

Membrane interaction and perturbation mechanisms induced by two cationic cell penetrating peptides with distinct charge distribution

Independently from the cell penetrating peptide uptake mechanism (endocytic or not), the interaction of the peptide with the lipid bilayer remains a common issue that needs further investigation. The cell penetrating or antimicrobial properties of exogenous peptides require probably different preliminary interactions with the plasma membrane. Herein, we have employed 31P NMR, differential scanning calorimetry and CD to study the membrane interaction and perturbation mechanisms of two basic peptides with similar length but distinct charge distribution, penetratin (non-amphipathic) and RL16, a secondary amphipathic peptide. The peptide effects on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dipalmitoleoyl phosphatidylethanolamine (DiPoPE) were investigated. We have found that, even though both peptides are cationic, their interaction with zwitterionic versus anionic lipids is markedly distinct. Penetratin greatly affects the temperature, enthalpy and cooperativity of DMPG main phase transition but does not affect those of DMPC while RL16 presents opposite effects. Additionally, it was found that penetratin induces a negative curvature whereas RL16 induces a positive one, since a decrease in the fluid lamellar to inverted hexagonal phase transition temperature of DiPoPE ( T H) was observed for penetratin and an increase for RL16. Contrary to penetratin, 31P NMR of samples containing DMPC MLVs and RL16 shows an isotropic signal indicative of the formation of small vesicles, concomitant with a great decrease in sample turbidity both below and at the phase transition temperature. Opposite effects were also observed on DMPG where both peptides provoke strong aggregation and precipitation. Both CPPs adopt helical structures when contacting with anionic lipids, and possess a dual behavior by either presenting their cationic or hydrophobic domains towards the phospholipid face, depending on the lipid nature (anionic vs zwitterionic, respectively). Surprisingly, the increase of electrostatic interactions at the water membrane interface prevents the insertion of RL16 hydrophobic region in the bilayer, but is essential for the interaction of penetratin. Modulation of amphipathic profiles and charge distribution of CPPs can alter the balance of hydrophobic and electrostatic membrane interaction leading to translocation or and membrane permeabilisation. Penetratin has a relative pure CPP behavior whereas RL16 presents mixed CPP/AMP properties. A better understanding of those processes is essential to unveil their cell translocation mechanism.

The Identification and Characterization of Fusogenic Domains in Herpes Virus Glycoprotein B Molecules

The molecular mechanism of entry of herpes viruses requires a multicomponent fusion system. Virus entry and cell-cell fusion of Herpes simplex virus (HSV) requires four glycoproteins: gD, gB and gH/gL. The role of gB remained elusive until recently, when the crystal structure of HSV-1 gB became available. Glycoprotein B homologues represent the most highly conserved group of herpes virus glycoproteins; however, despite the high degree of sequence and structural conservation, differences in post-translational processing are observed for different members of this virus family. Whereas gB of HSV is not proteolytically processed after oligomerization, most other gB homologues are cleaved by a cellular protease into subunits that remain linked through disulfide bonds. Proteolytic cleavage is common for activation of many other viral fusion proteins, so it remains difficult to envisage a common role for different herpes virus gB structures in the fusion mechanism. We selected bovine herpes virus type 1 (BoHV-1) and herpes simplex virus type 1 (HSV-1) as representative viruses expressing cleaved and uncleaved gBs, and have screened their amino acid sequences for regions of highly interfacial hydrophobicity. Synthetic peptides corresponding to such regions were tested for their ability to induce the fusion of large unilamellar vesicles and to inhibit herpes virus infection. These results underline that several regions of the gB protein are involved in the mechanism of membrane interaction.

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.

Biochemical characterization of the type I inositol polyphosphate 4-phosphatase C2 domain

Inositol polyphosphate 4 phosphatases (IP4Ps) are enzymes involved in the regulation of phosphoinositide 3-kinase lipid signaling. They catalyze the hydrolysis of the 4-position phosphate from phosphatidylinositol 3,4-bisphosphate to phosphatidylinositol 3-phosphate. In this paper we have characterized a lipid binding C2 domain located on the N-terminus of type I IP4Ps. Mutational analysis of the lipid binding loops suggests that Asp61, Asp120, Asp123, Lys122, Arg124 are involved in lipid binding in vitro. In addition, we show that this C2 domain binds calcium but calcium is not involved in lipid binding. This paper provides insight into the mechanism of membrane interaction of IP4Ps.

Hemolysis and antihemolysis induced by amino acid-based surfactants

Surfactants have the special ability to interact with the lipid bilayer of cell membranes. The red blood cell is one of the most used cellular membrane models to study the mechanisms underlying surfactant-induced osmotic cell resistance. To increase our knowledge regarding the mechanisms of surfactant membrane interaction, we studied the action of five lysine-derivative anionic and three arginine-derivative cationic amino acid-based surfactants on hypotonic hemolysis. Results showed two different antihemolytic behaviors among amino acid-based surfactants, both related to the maximal protective concentration. How the physico-chemical properties and structure of these compounds determine the protection against hypotonic hemolysis is discussed in detail. We found a good correlation between the CMC and the concentrations resulting in maximum protection against hypotonic hemolysis for the cationic surfactants, but no correlation for the anionic surfactants. In the case of lysine derivative surfactants, which only differ in their counterions, the counterion is implicated in the differences in the antihemolytic potency and the hemolytic activities of this.

Topography Studies on the Membrane Interaction Mechanism of the Eosinophil Cationic Protein

The eosinophil cationic protein (ECP) is an antipathogen protein involved in the host defense system. ECP displays bactericidal and membrane lytic capacities [Carreras et al. (2003) Biochemistry 42, 6636-6644]. We have now characterized in detail the protein-membrane interaction process. All observed fluorescent parameters of the wild type and single-tryptophan-containing mutants, as well as the results of decomposition analysis of protein fluorescence, suggest that W10 and W35 belong to two distinct spectral classes I and III, respectively. Tryptophan residues were classified and assigned to distinct structural classes using statistical approaches based on the analysis of tryptophan microenvironment structural properties. W10 belongs to class I and is buried in a relative nonpolar, nonflexible protein environment, while W35 (class III) is fully exposed to free water molecules. Tryptophan solvent exposure and the depth of the protein insertion in the lipid bilayer were monitored by the degree of protein fluorescence quenching by KI and brominated phospholipids, respectively. Results indicate that W35 partially inserts into the lipid bilayer, whereas W10 does not. Further analysis by electron microscopy and dynamic light scattering indicates that ECP can destabilize and trigger lipid vesicle aggregation at a nanomolar concentration range, corresponding to about 1:1000 protein/lipid ratio. No significant leakage of the vesicle aqueous content takes place below that protein concentration threshold. The data are consistent with a membrane destabilization "carpet-like" mechanism.