Peptide-lipid interactions of the β-hairpin antimicrobial peptide tachyplesin and its linear derivatives from solid-state NMR

The peptide-lipid interaction of a β-hairpin antimicrobial peptide tachyplesin-1 (TP-1) and its linear derivatives are investigated to gain insight into the mechanism of antimicrobial activity. ³¹P and ²H NMR spectra of uniaxially aligned lipid bilayers of varying compositions and peptide concentrations are measured to determine the peptide-induced orientational disorder and the selectivity of membrane disruption by tachyplesin. The disulfide-linked TP-1 does not cause any disorder to the neutral POPC and POPC/cholesterol membranes but induces both micellization and random orientation distribution to the anionic POPE/POPG membranes above a peptide concentration of 2%. In comparison, the anionic POPC/POPG bilayer is completely unaffected by TP-1 binding, suggesting that TP-1 induces negative curvature strain to the membrane as a mechanism of its action. Removal of the disulfide bonds by substitution of Cys residues with Tyr and Ala abolishes the micellization of POPE/POPG bilayers but retains the orientation randomization of both POPC/POPG and POPE/POPG bilayers. Thus, linear tachyplesin derivatives have membrane disruptive abilities but use different mechanisms from the wild-type peptide. The different lipid-peptide interactions between TP-1 and other β-hairpin antimicrobial peptides are discussed in terms of their molecular structure.     

Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843

Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH₂) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. ²H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. ³¹P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from ³¹P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and ³¹P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843.     

Amyloidogenic Propensities and Conformational Properties of ProIAPP and IAPP in the Presence of Lipid Bilayer Membranes

Human islet amyloid polypeptide (hIAPP), which is considered the primary culprit for β-cell loss in type 2 diabetes mellitus patients, is synthesized in β-cells of the pancreas from its precursor pro-islet amyloid polypeptide (proIAPP), which may be important in early intracellular amyloid formation as well. We compare the amyloidogenic propensities and conformational properties of proIAPP and hIAPP in the presence of negatively charged lipid membranes, which have been discussed as loci of initiation of the fibrillation reaction. Circular dichroism studies verify the initial secondary structures of proIAPP and hIAPP to be predominantly unordered with small amounts of ordered secondary structure elements, and exhibit minor differences between these two peptides only. Using attenuated total reflection-Fourier transform infrared spectroscopy and thioflavin T fluorescence spectroscopy, as well as atomic force microscopy, we show that in the presence of negatively charged membranes, proIAPP exhibits a much higher amyloidogenic propensity than in bulk solvent. Compared to hIAPP, it is still much less amyloidogenic, however. Although differences in the secondary structures of the aggregated species of hIAPP and proIAPP at the lipid interface are small, they are reflected in morphological changes. Unlike hIAPP, proIAPP forms essentially oligomeric-like structures at the lipid interface. Besides the interaction with anionic membranes [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) + x1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]], interaction with zwitterionic homogeneous (DOPC) and heterogeneous (1,2-dipalmitoyl-sn-glycero-3-phosphocholine:DOPC:cholesterol 1:2:1 model raft mixture) membranes has also been studied. Both peptides do not aggregate significantly at DOPC bilayers. In the presence of the model raft membrane, hIAPP aggregates markedly as well. Conversely, proIAPP clusters into less ordered structures and to a minor extent at raft membranes only. The addition of proIAPP to hIAPP retards the hIAPP fibrillation process also in the presence of negatively charged lipid bilayers. In excess proIAPP, increased aggregation levels are finally observed, however, which could be attributed to seed-induced cofibrillation of proIAPP.     

The effect of binding of spider-derived antimicrobial peptides, oxyopinins, on lipid membranes

Oxyopinins (Oxki1 and Oxki2) are antimicrobial peptides isolated from the crude venom of the wolf spider Oxyopes kitabensis. The effect of oxyopinins on lipid bilayers was investigated using high-sensitivity titration calorimetry and ³¹P solid-state NMR spectroscopy. High-sensitivity titration calorimetry experiments showed that the binding of oxyopinins was exothermic, and the binding enthalpies (ΔH) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) small unilamellar vesicles (SUVs) were − 18.1 kcal/mol and − 15.0 kcal/mol for Oxki1 and Oxki2, respectively, and peptide partition coefficient (K_p) was found to be 3.9 × 10³ M^− 1. ³¹P NMR spectra of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE) membranes in the presence of oxyopinins indicated that they induced a positive curvature in lipid bilayers. The induced positive curvature was stronger in the presence of Oxki2 than in the presence of Oxki1. ³¹P NMR spectra of phosphaditylcholine (PC) membranes in the presence of Oxki2 showed that Oxki2 produced micellization of membranes at low peptide concentrations, but unsaturated PC membranes or acidic phospholipids prevented micellization from occurring. Furthermore, ³¹P NMR spectra using membrane lipids from E. coli suggested that Oxki1 was more disruptive to bacterial membranes than Oxki2. These results strongly correlate to the known biological activity of the oxyopinins.     

Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides

Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the ¹⁵N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of ¹⁵N and ²H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.     

Reversible sheet-turn conformational change of a cell-penetrating peptide in lipid bilayers studied by solid-state NMR

The membrane-bound conformation of a cell-penetrating peptide, penetratin, is investigated using solid-state NMR spectroscopy. The ¹³C chemical shifts of ¹³C, ¹⁵N-labeled residues in the peptide indicate a reversible conformational change from β-sheet at low temperature to coil-like at high temperature. This conformational change occurs for all residues examined between positions 3 and 13, at peptide/lipid molar ratios of 1:15 and 1:30, in membranes with 25-50% anionic lipids, and in both saturated DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylchloline/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) membranes and unsaturated POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol) membranes. Thus, it is an intrinsic property of penetratin. The coil state of the peptide has C-H order parameters of 0.23-0.52 for C^α and C^β sites, indicating that the peptide backbone is unstructured. Moreover, chemical shift anisotropy lineshapes are uniaxially averaged, suggesting that the peptide backbone undergoes uniaxial rotation around the bilayer normal. These observations suggest that the dynamic state of penetratin at high temperature is a structured turn instead of an isotropic random coil. The thermodynamic parameters of this sheet-turn transition are extracted and compared to other membrane peptides reported to exhibit conformational changes. We suggest that the function of this turn conformation may be to reduce hydrophobic interactions with the lipid chains and facilitate penetratin translocation across the bilayer without causing permanent membrane damage.     

Influence of the lipid composition on the kinetics of concerted insertion and folding of melittin in bilayers

We have examined the kinetics of the adsorption of melittin, a secondary amphipathic peptide extracted from bee venom, on lipid membranes using three independent and complementary approaches. We probed (i) the change in the polarity of the ¹⁹Trp of the peptide upon binding, (ii) the insertion of this residue in the apolar core of the membrane, measuring the ¹⁹Trp-fluorescence quenching by bromine atoms attached on lipid acyl chains, and (iii) the folding of the peptide, by circular dichroism (CD). We report a tight coupling of the insertion of the peptide with its folding as an α-helix. For all the investigated membrane systems (cholesterol-containing, phosphoglycerol-containing, and pure phosphocholine bilayers), the decrease in the polarity of ¹⁹Trp was found to be significantly faster than the increase in the helical content of melittin. Therefore, from a kinetics point of view, the formation of the α-helix is a consequence of the insertion of melittin. The rate of melittin folding was found to be influenced by the lipid composition of the bilayer and we propose that this was achieved by the modulation of the kinetics of insertion. The study reports a clear example of the coupling existing between protein penetration and folding, an interconnection that must be considered in the general scheme of membrane protein folding.     

HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics

This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus K_C, a measure of membrane stiffness, and wide-angle x-ray scattering yielded the S_xray orientational order parameter. Both FP23 and FPtri decreased K_C and S_xray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion.     

Quantifying Interactions of β-Synuclein and γ-Synuclein with Model Membranes

The synucleins are a family of proteins involved in numerous neurodegenerative pathologies [α-synuclein and β-synuclein (βS)], as well as in various types of cancers [γ-synuclein (γS)]. While the connection between α-synuclein and Parkinson's disease is well established, recent evidence links point mutants of βS to dementia with Lewy bodies. Overexpression of γS has been associated with enhanced metastasis and cancer drug resistance. Despite their prevalence in such a variety of diseases, the native functions of the synucleins remain unclear. They have a lipid-binding motif in their N-terminal region, which suggests interactions with biological membranes in vivo. In this study, we used fluorescence correlation spectroscopy to monitor the binding properties of βS and γS to model membranes and to determine the free energy of the interactions. Our results show that the interactions are most strongly affected by the presence of both anionic lipids and bilayer curvature, while membrane fluidity plays a very minor role. Quantifying the lipid-binding properties of βS and γS provides additional insights into the underlying factors governing the protein-membrane interactions. Such insights not only are relevant to the native functions of these proteins but also highlight their contributions to pathological conditions that are either mediated or characterized by perturbations of these interactions.     

Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37

A 21-residue peptide segment, LL7-27 (RKSKEKIGKEFKRIVQRIKDF), corresponding to residues 7-27 of the only human cathelicidin antimicrobial peptide, LL37, is shown to exhibit potent activity against microbes (particularly Gram-positive bacteria) but not against erythrocytes. The structure, membrane orientation, and target membrane selectivity of LL7-27 are characterized by differential scanning calorimetry, fluorescence, circular dichroism, and NMR experiments. An anilinonaphthalene-8-sulfonic acid uptake assay reveals two distinct modes of Escherichia coli outer membrane perturbation elicited by LL37 and LL7-27. The circular dichroism results show that conformational transitions are mediated by lipid-specific interactions in the case of LL7-27, unlike LL37. It folds into an α-helical conformation upon binding to anionic (but not zwitterionic) vesicles, and also does not induce dye leakage from zwitterionic lipid vesicles. Differential scanning calorimetry thermograms show that LL7-27 is completely integrated with DMPC/DMPG (3:1) liposomes, but induces peptide-rich and peptide-poor domains in DMPC liposomes. ¹⁵N NMR experiments on mechanically aligned lipid bilayers suggest that, like the full-length peptide LL37, the peptide LL7-27 is oriented close to the bilayer surface, indicating a carpet-type mechanism of action for the peptide. ³¹P NMR spectra obtained from POPC/POPG (3:1) bilayers containing LL7-27 show substantial disruption of the lipid bilayer structure and agree with the peptide's ability to induce dye leakage from POPC/POPG (3:1) vesicles. Cholesterol is shown to suppress peptide-induced disorder in the lipid bilayer structure. These results explain the susceptibility of bacteria and the resistance of erythrocytes to LL7-27, and may have implications for the design of membrane-selective therapeutic agents.     

Lipid Polymorphism Induced by Surfactant Peptide SP-B1-25

Pulmonary surfactant protein B (SP-B) is an essential protein for lowering surface tension in the alveoli. SP-B_1-25, a peptide comprised of the N-terminal 25 amino-acid residues of SP-B, is known to retain much of the biological activity of SP-B. Circular dichroism has shown that when SP-B_1-25 interacts with negatively charged lipid vesicles, it contains significant helical structure for the lipid compositions and peptide/lipid ratios studied here. The effect of SP-B_1-25 on lipid organization and polymorphisms was investigated via DSC, dynamic light scattering, transmission electron microscopy, and solid-state NMR spectroscopy. At 1-3 mol% peptide and physiologic temperature, SP-B_1-25 partitions at the interface of negatively charged PC/PG lipid bilayers. In lipid mixtures containing 1-5 mol% peptide, the structure of SP-B_1-25 remains constant, but ²H and ³¹P NMR spectra show the presence of an isotropic lipid phase in exchange with the lamellar phase below the T_m of the lipids. This behavior is observed for both DPPC/POPG and POPC/POPG lipid mixtures as well as for both the PC and PG components of the mixtures. For 1-3 mol% SP-B_1-25, a return to a single lamellar phase above the lipid mixture T_m is observed, but for 5 mol% SP-B_1-25 a significant isotropic component is observed at physiologic temperatures for DPPC and exchange broadening is observed in ²H and ³¹P NMR spectra of the other lipid components in the two mixtures. DLS and TEM rule out the formation of micellar structures and suggest that SP-B_1-25 promotes the formation of a fluid isotropic phase. The ability of SP-B_1-25 to fuse lipid lamellae via this mechanism, particularly those enriched in DPPC, suggests a specific role for the highly conserved N-terminus of SP-B in the packing of lipid lamellae into surfactant lamellar bodies or in stabilizing multilayer structures at the air-liquid interface. Importantly, this behavior has not been seen for the other SP-B fragments of SP-B_8-25 and SP-B_59-80, indicating a critical role for the proline rich first seven amino acids in this protein.     

Coupling Molecular Dynamics Simulations with Experiments for the Rational Design of Indolicidin-Analogous Antimicrobial Peptides

Antimicrobial peptides (AMPs) have attracted much interest in recent years because of their potential use as new-generation antibiotics. Indolicidin (IL) is a 13-residue cationic AMP that is effective against a broad spectrum of bacteria, fungi, and even viruses. Unfortunately, its high hemolytic activity retards its clinical applications. In this study, we adopted molecular dynamics (MD) simulations as an aid toward the rational design of IL analogues exhibiting high antimicrobial activity but low hemolysis. We employed long-timescale, multi-trajectory all-atom MD simulations to investigate the interactions of the peptide IL with model membranes. The lipid bilayer formed by the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was chosen as the model erythrocyte membrane; lipid bilayers formed from a mixture of POPC and the negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were chosen to model bacterial membranes. MD simulations with a total simulation time of up to 4 μs revealed the mechanisms of the processes of IL adsorption onto and insertion into the membranes. The packing order of these lipid bilayers presumably correlated to the membrane stability upon IL adsorption and insertion. We used the degree of local membrane thinning and the reduction in the order parameter of the acyl chains of the lipids to characterize the membrane stability. The order of the mixed 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol/POPC lipid bilayer reduced significantly upon the adsorption of IL. On the other hand, although the order of the pure-POPC lipid bilayer was perturbed slightly during the adsorption stage, the value was reduced more dramatically upon the insertion of IL into the membrane's hydrophobic region. The results imply that enhancing IL adsorption on the microbial membrane may amplify its antimicrobial activity, while the degree of hemolysis may be reduced through inhibition of IL insertion into the hydrophobic region of the erythrocyte membrane. In addition, through simulations, we identified the amino acids that are most responsible for the adsorption onto or insertion into the two model membranes. Positive charges are critical to the peptide's adsorption, whereas the presence of hydrophobic Trp8 and Trp9 leads to its deeper insertion. Combining the hypothetical relationships between the membrane disordering and the antimicrobial and hemolytical activities with the simulated results, we designed three new IL-analogous peptides: IL-K7 (Pro7 → Lys), IL-F89 (Trp8 and Trp9 → Phe), and IL-K7F89 (Pro7 → Lys; Trp8 and Trp9 → Phe). The hemolytic activity of IL-F89 is considerably lower than that of IL, whereas the antimicrobial activity of IL-K7 is greatly enhanced. In particular, the de novo peptide IL-K7F89 exhibits higher antimicrobial activity against Escherichia coli; its hemolytic activity decreased to only 10% of that of IL. Our simulated and experimental results correlated well. This approach—coupling MD simulations with experimental design—is a useful strategy toward the rational design of AMPs for potential therapeutic use.     

Thermodynamics of the coil <==> beta-sheet transition in a membrane environment

Biologically important peptides such as the Alzheimer peptide Abeta(1-40) display a reversible random coil <==>beta-structure transition at anionic membrane surfaces. In contrast to the well-studied random coil left arrow over right arrow alpha-helix transition of amphipathic peptides, there is a dearth on information on the thermodynamic and kinetic parameters of the random coil left arrow over right arrow beta-structure transition. Here, we present a new method to quantitatively analyze the thermodynamic parameters of the membrane-induced beta-structure formation. We have used the model peptide (KIGAKI)(3) and eight analogues in which two adjacent amino acids were substituted by their d-enantiomers. The positions of the d,d pairs were shifted systematically along the three identical segments of the peptide chain. The beta-structure content of the peptides was measured in solution and when bound to anionic lipid membranes with circular dichroism spectroscopy. The thermodynamic binding parameters were determined with isothermal titration calorimetry and the binding isotherms were analysed by combining a surface partition equilibrium with the Gouy-Chapman theory. The thermodynamic parameters were found to be linearly correlated with the extent of beta-structure formation. beta-Structure formation at the membrane surface is characterized by an enthalpy change of DeltaH(beta)=-0.23 kcal/mol per residue, an entropy change of DeltaS(beta)=-0.24 cal/mol K residue and a free energy change of DeltaG(beta)=-0.15 kcal/mol residue. An increase in temperature induces an unfolding of beta-structure. The residual free energy of membrane-induced beta-structure formation is close to that of membrane-induced alpha-helix formation.     

Characterization of membrane properties of inositol phosphorylceramide

Inositol phosphorylceramides (IPCs) are a class of anionic sphingolipids with a single inositol-phosphate head group coupled to ceramide. IPCs and more complex glycosylated IPCs have been identified in fungi, plants and protozoa but not in mammals. IPCs have also been identified in detergent resistant membranes in several organisms. Here we report on the membrane properties of the saturated N-palmitoyl-IPC (P-IPC) in one component bilayers as well as in complex bilayers together with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. The membrane properties of P-IPC were shown to be affected by calcium. According to anisotropy changes reported by DPH, the gel-to-liquid transition temperature (T_m) of P-IPC was 48 °C. Addition of 5 mM CaCl₂ during vesicle preparation markedly increased the T_m (65 °C). According to fluorescence quenching experiments in complex lipid mixtures, P-IPC formed sterol containing domains in an otherwise fluid environment. The P-IPC containing domains melted at a lower temperature and appeared to contain less sterol as compared to domains containing N-palmitoyl-sphingomyelin. Calcium further reduced the sterol content of the ordered domains and also increased the thermal stability of the domains. Calcium also induced vesicle aggregation of unilamellar vesicles containing P-IPC, as was observed by 4D confocal microscopy and dynamic light scattering. We believe that IPCs and the calcium induced effects could be important in numerous membrane associated cellular processes such as membrane fusion and in membrane raft linked processes.     

Detergent-like properties of magainin antibiotic peptides: A P solid-state NMR spectroscopy study

³¹P solid-state NMR spectroscopy has been used to investigate the macroscopic phase behavior of phospholipid bilayers in the presence of increasing amounts of magainin antibiotic peptides. Addition of >1 mol% magainin 2 to gel-phase DMPC or liquid crystalline POPC membranes respectively, results in ³¹P NMR spectra that are characterized by the coexistence of isotropic signals and line shapes typical for phospholipid bilayers. The isotropic signal intensity is a function of temperature and peptide concentration. At peptide concentrations >4 mol% of the resulting phospholipid ³¹P NMR spectra are characteristic of magnetically oriented POPC bilayers suggesting the formation of small disk-like micelles or perforated sheets. In contrast, addition of magainin to acidic phospholipids results in homogenous bilayer-type ³¹P NMR spectra with reduced chemical shift anisotropies. The results presented are in good agreement with the interfacial insertion of magainin helices with an alignment parallel to the surface of the phospholipid bilayers. The resulting curvature strain results in detergent-like properties of the amphipathic helical peptides.     

The Role of the Lipid Bilayer in Tau Aggregation

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the β-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.     

Conformational changes of chicken liver bile acid-binding protein bound to anionic lipid membrane are coupled to the lipid phase transitions

Chicken liver bile acid-binding protein (L-BABP) binds to anionic lipid membranes by electrostatic interactions and acquires a partly folded state [Nolan, V., Perduca, M., Monaco, H., Maggio, B. and Montich, G. G. (2003) Biochim. Biophys. Acta 1611, 98-106]. We studied the infrared amide I′ band of L-BABP bound to dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol (DMPG) and palmitoyloleoylphosphatidylglycerol (POPG) in the range of 7 to 60 °C. Besides, the thermotrophic behaviour of DPPG and DMPG was studied in the absence and in the presence of bound-protein by differential scanning calorimetry (DSC) and infrared spectra of the stretching vibration of methylene and carbonyl groups. When L-BABP was bound to lipid membranes in the liquid-crystalline state (POPG between 7 and 30 °C) acquired a more unfolded conformation that in membranes in the gel state (DPPG between 7 and 30 °C). Nevertheless, this conformational change of the protein in DMPG did not occur at the temperature of the lipid gel to liquid-crystalline phase transition detected by infrared spectroscopy. Instead, the degree of unfolding in the protein was coincident with a phase transition in DMPG that occurs with heat absorption and without change in the lipid order.     

Structure analysis of the membrane protein TatCd from the Tat system of B. subtilis by circular dichroism

The twin arginine translocation (Tat) system can transport fully folded proteins, including their cofactors, across bacterial and thylakoid membranes. The Tat system of Bacillus subtilis that serves to export the phosphodiesterase (PhoD) consists of only two membrane proteins, TatA_d and TatC_d. The larger component TatC_d has a molecular weight of 28 kDa and several membrane-spanning segments. This protein has been expressed in Escherichia coli and purified in sufficient amounts for structure analysis by circular dichroism (CD) and NMR spectroscopy. TatC_d was reconstituted in detergent micelles and in lipid bilayers for CD analysis in solution and in macroscopically oriented samples, to examine the stability of the protein. Suitable protocols and model membrane systems have been established, by which TatC_d maintains the level of helicity close to theoretically predicted, and its transmembrane alignment could been verified.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.