Penetratin-membrane association: W48/R52/W56 shield the peptide from the aqueous phase

Using molecular dynamics simulations, we studied the mode of association of the cell-penetrating peptide penetratin with both a neutral and a charged bilayer. The results show that the initial peptide-lipid association is a fast process driven by electrostatic interactions. The homogeneous distribution of positively charged residues along the axis of the helical peptide, and especially residues K46, R53, and K57, contribute to the association of the peptide with lipids. The bilayer enhances the stability of the penetratin helix. Oriented parallel to the lipid-water interface, the subsequent insertion of the peptide through the bilayer headgroups is significantly slower. The presence of negatively charged lipids considerably enhances peptide binding. Lateral side-chain motion creates an opening for the helix into the hydrophobic core of the membrane. The peptide aromatic residues form a pi-stacking cluster through W48/R52/W56 and F49/R53, protecting the peptide from the water phase. Interaction with the penetratin peptide has only limited effect on the overall membrane structure, as it affects mainly the conformation of the lipids which interact directly with the peptide. Charge matching locally increases the concentration of negatively charged lipids, lateral lipid diffusion locally decreases. Lipid disorder increases, through decreased order parameters of the lipids interacting with the penetratin side chains. Penetratin molecules at the membrane surface do not seem to aggregate.     

A critical reassessment of penetratin translocation across lipid membranes

Penetratin is a short, basic cell-penetrating peptide able to induce cellular uptake of a vast variety of large, hydrophilic cargos. We have reassessed the highly controversial issue of direct permeation of the strongly cationic peptide across negatively charged lipid membranes. Confocal laser scanning microscopy on rhodamine-labeled giant vesicles incubated with carboxyfluorescein-labeled penetratin yielded no evidence of transbilayer movement, in contradiction to previously reported results. Confocal fluorescence spectroscopy on black lipid membranes confirmed this finding, which was also not affected by application of a transmembrane electric potential difference. A novel dialysis assay based on tryptophan absorbance and fluorescence spectroscopy demonstrated that the permeability of small and large unilamellar vesicles to penetratin is <10(-13) m/s. Taken together, the results show that penetratin is not capable of overcoming model membrane systems irrespective of the bilayer curvature or the presence of a transmembrane voltage. Thus, direct translocation across the hydrophobic core of the plasma membrane cannot account for the efficient uptake of penetratin into live cells, which is in accord with recent in vitro studies underlining the importance of endocytosis in the internalization process of cationic cell-penetrating peptides.     

Kinetics of melittin binding to phospholipid small unilamellar vesicles

We have used the decrease in the fluorescence intensity of the single tryptophan residue of bee venom melittin at long emission wavelengths that accompanies binding of the peptide to phospholipid small unilamellar vesicles to determine the rate of binding through the use of stopped-flow fluorometry in the millisecond range. We have found the rate to depend on the degree of saturation of the lipid acyl chains as well as on the physical state of the bilayer, the net electric charge of the polar headgroups, and the lipid-to-melittin molar ratio R. For zwitterionic lipids (i) the binding process is found to exhibit negative cooperativity, and (ii) the rate-limiting step appears to be penetration of the protein into the hydrophobic region of the bilayer. For negatively charged lipids the results show that binding is a very fast process that seems to be electrostatic in nature.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

Relationships between membrane binding, affinity and cell internalization efficacy of a cell-penetrating peptide: penetratin as a case study

Background

Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.

Methodology

Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG^neg) and sialic acid-deficient (SA^neg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.

Principal Findings

Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA^neg>WT>GAG^neg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA^neg and GAG^neg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG^neg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.

Conclusion/Significance

These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering.     

Application of a novel analysis to measure the binding of the membrane-translocating peptide penetratin to negatively charged liposomes

The binding of penetratin, a peptide that has been found useful for cellular delivery of large hydrophilic molecules, to negatively charged vesicles was investigated. The surface charge density of the vesicles was varied by mixing zwitterionic dioleoylphosphatidylcholine (DOPC) and negatively charged dioleoylphosphatidylglycerol (DOPG) at various molar ratios. The extent of membrane association was quantified from tryptophan emission spectra recorded during titration of peptide solution with liposomes. A singular value decomposition of the spectral data demonstrated unambiguously that two species, assigned as peptide free in solution and membrane-bound peptide, respectively, account for the spectral data of the titration series. Binding isotherms were then constructed by least-squares projection of the titration spectra on reference spectra of free and membrane-bound peptide. A model based on the Gouy-Chapman theory in combination with a two-state surface partition equilibrium, separating the electrostatic and the hydrophobic contributions to the binding free energy, was found to be in excellent agreement with the experimental data. Using this model, a surface partition constant of approximately 80 M(-)(1) was obtained for the nonelectrostatic contribution to the binding of penetratin irrespective of the fraction of negatively charged lipids in the membrane, indicating that the hydrophobic interactions are independent of the surface charge density. In accordance with this, circular dichroism measurements showed that the secondary structure of membrane-associated penetratin is independent of the DOPC/DOPG ratio. Experiments using vesicles with entrapped carboxyfluorescein showed that penetratin does not form membrane pores. Studies of the cationic peptide penetratin are complicated by extensive adsorption to surfaces of quartz and plastics. By modification of the quartz cell walls with the cationic polymer poly(ethylenimine), the peptide adsorption was reduced to a tolerable level. The data analysis method used for construction of the binding isotherms eliminated errors emanating from the remaining peptide adsorption, which otherwise would prevent a proper quantification of the binding.     

Membrane interaction and cellular internalization of penetratin peptides.

Penetratin is a 16-residue peptide [RQIKIWFQNRRMKWKK(43-58)] derived from the Antennapedia homeodomain, which is used as a vector for cellular internalization of hydrophilic molecules. In order to unravel the membrane translocation mechanism, we synthesized new penetratin variants. The contribution of the positively charged residues was studied by double substitutions of Lys and/or Arg residues to Ala, while the specific contribution of Trp48 and Trp56 was studied by individual substitution of these residues to Phe. Trp fluorescence titrations demonstrated the importance of the positively charged residues for the initial electrostatic interaction of the peptide with negatively charged vesicles. In contrast, none of the Trp residues seemed critical for this initial interaction. Trp fluorescence quenching experiments showed that penetratin lies close to the water-lipid interface in a tilted orientation, while circular dichroism indicated that lipid binding increased the alpha-helical structure of the peptides. The R53A/K57A and R52A/K55A substitutions increased calcein leakage and decreased vesicle aggregation compared to wild-type penetratin. These variants insert deeper into the lipid bilayer, due to an increased hydrophobic environment of Trp56. The W48F and W56F substitutions had a minor effect on membrane insertion and destabilization. Cellular internalization of the R53A/K57A, R52A/K55A and K46A/K57A variants by MDCK cells was similar to wild-type penetratin, as shown by flow cytometry. Moreover, residue Trp48 specifically contributed to endocytosis-independent internalization by MDCK cells, as demonstrated by the lower uptake of the W48F variant compared to wild-type penetratin and to the W56F variant. None of the penetratin variants was haemolytic or cytotoxic.     

A molecular view on the interaction of the trojan peptide penetratin with the polar interface of lipid bilayers

Penetratin belongs to the family of Trojan peptides that effectively enter cells and therefore can be used as cargoes for agents that are unable to penetrate the cell membrane. We applied polarized infrared spectroscopy in combination with the attenuated total reflection technique to extract information before penetratin binding to lipid membranes with molecular resolution. The amide I band of penetratin in the presence of zwitterionic dimyristoylphosphatidylcholine and of anionic lipid membranes composed of dioleoylphosphatidylcholine and dioleoylphosphatidylglycerol shows the characteristics of an antiparallel beta-sheet with a small fraction of turns. Both signatures have been interpreted in terms of a hairpin conformation. The infrared linear dichroism of the amide I band indicates that the peptide chain orients in an oblique fashion whereas the plane of the sheet aligns virtually parallel with respect to the membrane surface. The weak effect of the peptide on dimyristoylphosphatidylcholine gives indication of its superficial binding where the charged lysine and arginine side chains form H-bonds to the phosphate oxygens of the surrounding lipids. The determinants for internalization of penetratin appear to be a peptide sequence with a distribution of positively charged residues along a beta-sheet conformation, which enables the anchoring of the peptide in the polar part of the membranes and the effective compensation of anionic lipid charges.     

The interaction of the cell-penetrating peptide penetratin with heparin, heparansulfates and phospholipid vesicles investigated by ESR spectroscopy.

An ESR investigation of the interaction of spin-labelled penetratin with heparin, heparansulfates and several phospholipid vesicle formulations is reported. Penetratin is a 16-aa peptide corresponding to the third helix of the Antennapedia homeodomain and belonging to the cell-penetrating peptide family. The present study shows that ESR spectroscopy can provide specific and reliable information about the mechanism of interaction of penetratin with polysaccharides and lipids, at a molecular level. The study showed that: (i) heparin and heparansulfates specifically interact with spin-labelled penetratin and promote peptide aggregation and concentration on their molecular surface; (ii) penetratin does not interact with neutral lipids, whereas it enters negatively charged lipid bilayers; (iii) cholesterol plays a negative effect on the insertion of penetratin into the lipid membrane; (iv) the interaction of penetratin with lipid vesicles is strongly dependent on lipid concentration. In a low lipid regime, penetratin associates with the polar heads of phospholipids and aggregates on the membrane surface; once the lipid concentration attains a threshold, the peptide enters the lipid bilayer. This step is characterized by reduced peptide mobility and partial disaggregation.It has been shown that ESR spectroscopy is a valuable investigation tool in studies related to the still unclear mechanism of the internalization process.     

Modulation of melittin-induced lysis by surface charge density of membranes.

Phosphorus NMR spectroscopy was used to characterize the importance of electrostatic interactions in the lytic activity of melittin, a cationic peptide. The micellization induced by melittin has been characterized for several lipid mixtures composed of saturated phosphatidylcholine (PC) and a limited amount of charged lipid. For these systems, the thermal polymorphism is similar to the one observed for pure PC: small comicelles are stable in the gel phase and extended bilayers are formed in the liquid crystalline phase. Vesicle surface charge density influences strongly the micellization. Our results show that the presence of negatively charged lipids (phospholipid or unprotonated fatty acid) reduces the proportion of lysed vesicles. Conversely, the presence of positively charged lipids leads to a promotion of the lytic activity of the peptide. The modulation of the lytic effect is proposed to originate from the electrostatic interactions between the peptide and the bilayer surface. Attractive interactions anchor the peptide at the surface and, as a consequence, inhibit its lytic activity. Conversely, repulsive interactions favor the redistribution of melittin into the bilayer, causing enhanced lysis. A quantitative analysis of the interaction between melittin and negatively charged bilayers suggests that electroneutrality is reached at the surface, before micellization. The surface charge density of the lipid layer appears to be a determining factor for the lipid/peptide stoichiometry of the comicelles; a decrease in the lipid/peptide stoichiometry in the presence of negatively charged lipids appears to be a general consequence of the higher affinity of melittin for these membranes.     

Binding of prion protein to lipid membranes and implications for prion conversion

The binding of the Syrian hamster prion protein, SHaPrP(90-231), to model lipid membranes was investigated by tryptophan fluorescence. Membranes composed of negatively charged or zwitterionic lipids, and raft-like membranes containing dipalmitoylphosphatidylcholine(1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol and sphingomyelin, were investigated. It was found that SHaPrP(90-231) binds to negatively charged lipid membranes and raft-like membranes. Binding of PrP to negatively charged lipid membranes involves both electrostatic and hydrophobic lipid-protein interactions and results in partial insertion of PrP into the lipid bilayer. This membrane-inserted conformation of PrP is richer in beta-sheet structure and has a disruptive effect on the integrity of the lipid bilayer, leading to total release of vesicle contents. In contrast, the binding of PrP to raft-like membranes is driven by hydrophobic lipid-protein interactions and induces the formation of alpha-helical structure. This conformation of PrP with a high content of alpha-helix is formed only at pH 7 and does not destabilize the lipid bilayer. Our findings support the view that an interaction of PrP with lipid membranes could play a role in PrP conversion.     

Tryptophan fluorescence study on the interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with model membranes

The interaction of the signal peptide of the Escherichia coli outer membrane protein PhoE with different phospholipid vesicles was investigated by fluorescence techniques, using a synthetic mutant signal peptide in which valine at position -8 in the hydrophobic sequence was replaced by tryptophan. First it was established that this mutation in the signal sequence of prePhoE does not affect in vivo and in vitro translocation efficiency and that the biophysical properties of the synthetic mutant signal peptide are similar to those of the wild-type signal peptide. Next, fluorescence experiments were performed which showed an increase in quantum yield and a blue shift of the emission wavelength maximum upon interaction of the signal peptide with lipid vesicles, indicating that the tryptophan moiety enters a more hydrophobic environment. These changes in intrinsic fluorescence were found to be more pronounced in the presence of phosphatidylglycerol (PG) or cardiolipin (CL) than with phosphatidylcholine (PC). In addition, quenching experiments demonstrated a shielding of the tryptophan fluorescence from quenching by the aqueous quenchers iodide and acrylamide upon interaction of the signal peptide with lipid vesicles, a shielding in the case of acrylamide that was more pronounced in the presence of negatively charged lipids. Finally it was found that acyl chain brominated lipids incorporated into phospholipid bilayers were able to quench the tryptophan fluorescence of the signal peptide, with the quenching efficiency in CL vesicles being much higher than in PC vesicles. The results clearly demonstrate that the PhoE signal peptide interacts strongly with different lipid vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of cationic pentapeptides with modified side chain lengths to negatively charged lipid membranes: Complex interplay of electrostatic and hydrophobic interactions

Basic amino acids play a key role in the binding of membrane associated proteins to negatively charged membranes. However, side chains of basic amino acids like lysine do not only provide a positive charge, but also a flexible hydrocarbon spacer that enables hydrophobic interactions. We studied the influence of hydrophobic contributions to the binding by varying the side chain length of pentapeptides with ammonium groups starting with lysine to lysine analogs with shorter side chains, namely ornithine (Orn), α,γ-diaminobutyric acid (Dab) and α, β-diaminopropionic acid (Dap). The binding to negatively charged phosphatidylglycerol (PG) membranes was investigated by calorimetry, FT-infrared spectroscopy (FT-IR) and monolayer techniques. The binding was influenced by counteracting and sometimes compensating contributions. The influence of the bound peptides on the lipid phase behavior depends on the length of the peptide side chains. Isothermal titration calorimetry (ITC) experiments showed exothermic and endothermic effects compensating to a different extent as a function of side chain length. The increase in lipid phase transition temperature was more significant for peptides with shorter side chains. FTIR-spectroscopy revealed changes in hydration of the lipid bilayer interface after peptide binding. Using monolayer techniques, the contributions of electrostatic and hydrophobic effects could clearly be observed. Peptides with short side chains induced a pronounced decrease in surface pressure of PG monolayers whereas peptides with additional hydrophobic interactions decreased the surface pressure much less or even lead to an increase, indicating insertion of the hydrophobic part of the side chain into the lipid monolayer.     

Membrane binding mechanism of an RNA virus-capping enzyme

The RNA replication complex of Semliki Forest virus is bound to cytoplasmic membranes via the mRNA-capping enzyme Nsp1. Here we have studied the structure and liposome interactions of a synthetic peptide (245)GSTLYTESRKLLRSWHLPSV(264) corresponding to the membrane binding domain of Nsp1. The peptide interacted with liposomes only if negatively charged lipids were present that induced a structural change in the peptide from a random coil to a partially alpha-helical conformation. NMR structure shows that the alpha-helix is amphipathic, the hydrophobic surface consisting of several leucines, a valine, and a tryptophan moiety (Trp-259). Fluorescence studies revealed that this tryptophan intercalates in the bilayer to the depth of the ninth and tenth carbons of lipid acyl chains. Mutation W259A altered the mode of bilayer association of the peptide and abolished its ability to compete for membrane association of intact Nsp1, demonstrating its crucial role in the membrane association and function of Nsp1.     

Effect of charged residue substitutions on the thermodynamics of signal peptide-lipid interactions for the Escherichia coli LamB signal sequence.

We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under approximately physiological ionic strength. In addition, the lipid affinities of model surface-binding and transmembrane peptides were determined. These comparative studies with signal and model peptides permitted semi-quantitative deconvolution of signal peptide binding into electrostatic and hydrophobic components. We find that both interactions contribute significantly to binding, although the theoretically available hydrophobic free energy is largely offset by unfavorable polar-group effects. The implications of these results for understanding the potential roles of the signal sequence in protein translocation are discussed.     

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.     

The role of charge and hydrophobicity in peptide-lipid interaction: a comparative study based on tryptophan fluorescence measurements combined with the use of aqueous and hydrophobic quenchers

The interaction of interrelated model peptides with model membranes has been studied by techniques based on tryptophan fluorescence. The peptides used are derivatives of the sequence H-Ala-Met-Leu-Trp-Ala-OH, which was designed for this purpose. Several modifications yielded a set of 13 penta- and hexapeptides varying in net charge, hydrophobicity, charge distribution, and the intramolecular position of the tryptophan residue with respect to the charge(s). The affinity of these peptides for small unilamellar vesicles (SUV) consisting of zwitterionic egg phosphatidylcholine (eggPC) and negatively charged beef heart cardiolipin (bhCL) has been investigated in a comparative way. The criteria for affinity comprise (1) intrinsic fluorescence changes upon titration of the peptides with the lipid vesicles, (2) reduced accessibility of the peptides to aqueous quenchers of tryptophan fluorescence (I- and acrylamide) in the presence of lipid, and (3) exposure to membrane-incorporated fluorescence quenchers, brominated phosphatidylcholines (BrPC). Application of BrPC brominated at different positions along the acyl chains provided information on the membrane topology of the peptides. With respect to the extent of affinity for zwitterionic membranes, the overall hydrophobicity of the peptides is the main determinant. A comparison of the affinity for PC of equally hydrophobic peptides carrying either a single positive or negative charge reveals preferential interaction of the cationic peptide. Both hydrophobic and electrostatic interactions determine the affinity of positively charged mono- and divalent peptides for CL vesicles. The distribution of the charged moieties in divalent positively charged peptides, either both at one end of the molecule or one at each end, has little influence on the affinity of these peptides for CL but does affect the extent of exposure to BrPC. Upon decreasing the surface charge density of the vesicles by diluting CL with increasing amounts of PC, both types of peptides show different behavior. The position of the tryptophan relative to the charged moiety in the peptide molecule is shown to affect the fluorescent properties upon interaction with vesicles. Concerning the membrane topology, all peptides adopt a localization near the membrane surface, with the neutral peptides inserting slightly deeper into the bilayer than the charged peptides. The results allow a comparative analysis of the factors determining the extents and modes of lipid-model peptide interaction; in addition, the validity of the methods applied is discussed.     

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 (31)P 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, (31)P 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.     

Protein transduction domains of HIV-1 and SIV TAT interact with charged lipid vesicles. Binding mechanism and thermodynamic analysis

Cell-penetrating peptides (CPPs) traverse cell membranes of cultured cells very efficiently by a mechanism not yet identified. Recent theories for the translocation suggest either the binding of the CPPs to extracellular glycosaminoglycans or the formation of inverted micelles with negatively charged lipids. In the present study, the binding of the protein transduction domains (PTD) of human (HIV-1) and simian immunodeficiency virus (SIV) TAT peptide (amino acid residues 47-57, electric charge z(p) = +8) to membranes containing various proportions of negatively charged lipid (POPG) is characterized. Monolayer expansion measurements demonstrate that TAT-PTD insertion between lipids requires loosely packed monolayer films. For densely packed monolayers (pi > 29 mN/m) and lipid bilayers, no insertion is possible, and binding occurs via electrostatic adsorption to the membrane surface. Light scattering experiments show an aggregation of anionic lipid vesicles when the electric surface charge is neutralized by TAT-PTD, the observed stoichiometry being close to the theoretical value of 1:8. Membrane binding was quantitated with isothermal titration calorimetry and three further methods. The reaction enthalpy is Delta H degrees approximately equal to -1.5 kcal/mol peptide and is almost temperature-independent with Delta C(p) degrees approximately 0 kcal/(mol K), indicating equal contributions of polar and hydrophobic interactions to the reaction heat capacity. The binding of TAT-PTD to the anionic membrane is described by an electrostatic attraction/chemical partition model. The electrostatic attraction energy, calculated with the Gouy-Chapman theory, accounts for approximately 80% of the binding energy. The overall binding constant, K(app), is approximately 10(3)-10(4) M(-1). The intrinsic binding constant (K(p)), corrected for electrostatic effects and describing the partitioning of the peptide between the lipid-water interface and the membrane, is small and is K(p) approximately 1-10 M(-1). Deuterium and phosphorus-31 nuclear magnetic resonance demonstrate that the lipid bilayer remains intact upon TAT-PTD binding. The NMR data provide no evidence for nonbilayer structures and also not for domain formation. This is further supported by the absence of dye efflux from single-walled lipid vesicles. The electrostatic interaction between TAT-PTD and anionic phosphatidylglycerol is strong enough to induce a change in the headgroup conformation of the anionic lipid, indicating a short-lived but distinct correlation between the TAT-PTD and the anionic lipids on the membrane outside. TAT-PTD has a much lower affinity for lipid membranes than for glycosaminoglycans, making the latter interaction a more probable pathway for CPP binding to biological membranes.