Structure, orientation and affinity for interfaces and lipids of ideally amphipathic lytic LiKj(i=2j) peptides

The behavior of lytic ideally amphipathic peptides of generic composition LiKj(i=2j) and named LKn, n=i+j, is investigated in situ by the monolayer technique combined with the recently developed polarization modulation IR spectroscopy (PMIRRAS). A change in the secondary structure occurs versus peptide length. Peptides longer than 12 residues fold into alpha-helices at interfaces as expected from their design, while enough shorter peptides, from 9 down to 5 residues, form intermolecular antiparallel beta-sheets. Analysis of experimental and calculated PMIRRAS spectra in the amide I and II regions show that peptides are flat oriented at the interfaces. Structures and orientation are preserved whatever the nature of the interface, air/water or DMPC monolayer, and the lateral pressure. Peptide partition constants, KaffPi, are estimated from isobar surface increases of DMPC monolayers. They strongly increase when Pi decreases from 30 mN/m to 8 mN/m and they vary with peptide length with an optimum for 12 residues. This non-monotonous dependence fits with data obtained in bilayers and follows the hemolytic activity of the peptides. Lipid perturbations due to peptide insertion essentially detected on the PO4- and CO bands indicate disorder of the lipid head groups. Lysis induced on membranes by such peptides is proposed to first result from their flat asymmetric insertion.     

Contribution of the hydrophobicity gradient of an amphipathic peptide to its mode of association with lipids

A class of peptides that associate with lipids, known as oblique-orientated peptides, was recently described [Brasseur R., Pillot, T., Lins, L., Vandekerckhove, J. & Rosseneu, M. (1997) Trends Biochem. Sci. 22, 167-171]. Due to an asymmetric distribution of hydrophobic residues along the axis of the alpha-helix, such peptides adopt an oblique orientation which can destabilise membranes or lipid cores. Variants of these oblique peptides, designed to have an homogeneous distribution of hydrophobic and hydrophilic residues along the helical axis, are classified as regular amphipathic peptides. These peptides are expected to lie parallel to the polar/apolar interface with their hydrophobic residues directed towards the apolar and their hydrophilic residues towards the polar phase. An hydrophobic, oblique-orientated peptide was identified at residues 56-68 in the sequence of the lecithin-cholesterol acyltransferase (LCAT), enzyme. This peptide is predicted to penetrate a lipid bilayer at an angle of 40 degrees through its more hydrophobic C-terminal end and thereby induce the destabilisation of a membrane or a lipid core. The LCAT-(56-68) wild-type peptide was synthesised together with the LCAT-(56-68, 0 degrees) variant, in which the hydrophobicity gradient was abolished through residue permutations. In two other variants, designed to keep their oblique orientation, the W61 residue was shifted either towards the more hydrophilic N-terminal at residue 57, or to position 68 at the hydrophobic C-terminal end of the peptide. Peptide-induced vesicle fusion was demonstrated by fluorescence measurements using pyrene-labeled vesicles and by monitoring of vesicle size by gel filtration. The interaction between peptides and lipids was monitored by measurement of the intrinsic tryptophan fluorescence emission of the peptides. Fluorescence polarisation measurements, using diphenyl hexatriene, were carried out to follow changes in the lipid fluidity. The LCAT-(56-68) wild-type peptide and the two oblique variants, induced fusion of unilamellar dimyristoylglycerophosphocholine vesicles. Tryptophan fluorescence emission measurements showed a 12-14 nm blue shift upon addition of the wild-type peptide and of the W61-->68 variant to lipids, whereas the fluorescence of the W61-->57 variant did not change significantly. This observation supports the insertion of the more hydrophobic C-terminal residues into the lipid phase, as predicted by the theoretical calculations. In contrast, the 0 degrees variant peptide had no fusogenic activity, and it associated with lipids to form small discoidal lipid/peptide complexes. The phospholipid transition temperature was decreased after addition of the wild-type, the W61-->68 and W61-->57 fusogenic peptides, whereas the opposite effect was observed with the 0 degrees variant. The behaviour of the wild-type and variant LCAT-(56-68) peptides stresses the contribution of the hydrophobicity gradient along the axis of an amphipathic peptide to the mode of association of this peptide with lipids. This parameter consequently influences the structural modifications occurring to lipids upon association with amphipathic peptides.     

A physicochemical approach for predicting the effectiveness of peptide-based gene delivery systems for use in plasmid-based gene therapy

Novel synthetic peptides, based on carrier peptide analogs (YKAKnWK) and an amphipathic peptide (GLFEALLELLESLWELLLEA), have been formulated with DNA plasmids to create peptide-based gene delivery systems. The carrier peptides are used to condense plasmids into nanoparticles with a hydrodynamic diameter (DH) ranging from 40 to 200 nm, which are sterically stable for over 100 h. Size and morphology of the carrier peptide/plasmid complex have been determined by photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM), respectively. The amphipathic peptide is used as a pH-sensitive lytic agent to facilitate release of the plasmid from endosomes after endocytosis of the peptide/plasmid complex. Hemolysis assays have shown that the amphipathic peptide destabilizes lipid bilayers at low pH, mimicking the properties of viral fusogenic peptides. However, circular dichroism studies show that unlike the viral fusion peptides, this amphipathic peptide loses some of its alpha-helical structure at low pH in the presence of liposomes. The peptide-based gene delivery systems were tested for transfection efficiency in a variety of cell lines, including 14-day C2C12 mouse myotubes, using gene expression systems containing the beta-galactosidase reporter gene. Transfection data demonstrate a correlation between in vitro transfection efficiency and the combination of several physical properties of the peptide/plasmid complexes, including 1) DNA dose, 2) the zeta potential of the particle, 3) the requirement of both lytic and carrier peptides, and 4) the number of lysine residues associated with the carrier peptide. Transfection data on 14-day C2C12 myotubes utilizing the therapeutic human growth hormone gene formulated in an optimal peptide gene delivery system show an increase in gene expression over time, with a maximum in protein levels at 96 h (approximately 18 ng/ml).     

A mathematically defined motif for the radial distribution of charged residues on apolipoprotein amphipathic alpha helixes

Multiple amphipathic alpha-helical candidate domains have been identified in exchangeable apolipoproteins by sequence analysis and indirect experimental evidence. The distribution of charged residues can differ within and between these apolipoproteins. Segrest et al. (Segrest, J. P., H. DeLoof, J. G. Dohlman, C. G. Brouillette, and G. M. Anantharamaiah. 1990. Proteins. 8:103-117.) argued that these differences are correlated with lipid affinity. A mathematically defined motif for the particular charge distribution associated with high lipid affinity (class A) is proposed. Primary sequence data from protein segments proposed previously to have an amphipathic alpha-helical structure are scanned. Counting formulas are presented for determining the conditional probability that the match between an observed charge distribution and the proposed motif would occur by chance. Because the preselected helical segments are short (the modal length is 22) and the motif definition imposes multiple constraints on the acceptable distributions, the computer-based algorithm is quite feasible computationally. 19 of the 20 segments previously assigned to class A match the motif sufficiently well (the remaining one is borderline), while very few others "erroneously" pass the screening test. These results confirm the original assignments of the candidate domains and, thus, support the hypothesis that there is a distinguishable subset of helixes having high lipid affinity. This counting approach is applicable to a growing subset of protein sequence analysis problems in which the segment lengths are short and the motif is complex.     

Conformation and lipid binding properties of four peptides derived from the membrane-binding domain of CTP:phosphocholine cytidylyltransferase

We are probing the mechanism of the lipid selective membrane interactions of CTP:phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic alpha-helix between residues approximately 240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as alpha-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only approximately 1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule).     

Role of lipids in the permeabilization of membranes by class L amphipathic helical peptides

We studied the mechanism of membrane permeabilization by the 18L model peptide (GIKKFLGSIWKFIKAFVG), which features the consensus class L sequence averaged from the number of naturally occurring lytic peptides. Two aspects of membrane lipid composition significantly affected peptide-membrane interactions: the presence of acidic lipids and, in zwitterionic membranes, and the presence of nonbilayer forming lipids. In zwitterionic membranes, 18L peptide destabilizes the membrane, leading to a transient formation of large defects in the membrane which result generally in contents leakage, but in the presence of bilayer-bilayer contact can alternatively lead to vesicle fusion. In membranes containing acidic lipids (DOPC:DOPG, DOPG), 18L caused leakage but not fusion, probably due to mutual repulsion of acidic vesicles. While the extent of contents leakage was approximately the same as for zwitterionic membranes, the kinetics of leakage could be resolved only by using stopped-flow, leakage being essentially complete within the first minute. Previously, we reported that apolipoprotein (class A) and lytic (class L) peptide analogs have opposing effects on some properties of biological membranes. This reciprocal effect of 18L and Ac-18A-NH2, class A model peptide, is restricted to membranes with a high propensity for nonbilayer phase formation (DOPE, Me-DOPE, DOPC:DOPE, DOPC:Me-DOPE). The decrease in the content of nonbilayer phase forming lipid or the addition of acidic lipids reduces or eliminates the reciprocal effects. This suggests the importance of nonbilayer phase propensity for certain functions of biological membranes.     

Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence, monolayer and modeling

A cationic amphiphilic peptide made of 10 leucine and 10 lysine residues, and four of its fluorescent derivatives in which leucines were substituted by Trp residues at different locations on the primary sequence have been synthesized. The interactions of these five peptides with neutral anionic or cationic vesicles were investigated using circular dichroism, steady state and time-resolved fluorescence with a combination of Trp quenching by brominated lipid probes, monolayers, modeling with minimization and simulated annealing procedures. We show that all the five peptides interact with neutral and anionic DMPC, DMPG, DOPC or egg yolk PC vesicles. The binding takes place whatever the peptide conformation in solution is. In the case of DMPC bilayers the binding free energy DeltaG is estimated at -8 kcal mole-1 and the number of phospholipid molecules involved is about 20-25 per peptide molecule. Peptides are bound as single-stranded alpha helices orientated parallel to the bilayer surface. In the anchoring of phospholipid head groups around the peptides, the lipid molecules are not smeared out in a plane parallel to the membrane surface but are organized around the hydrophilic face of the alpha helices like 'wheat grains around an ear' and protrude outside the bilayer towards the solvent. We suggest that such a lipid arrangement generates transient structural defects responsible for the membrane permeability enhancement. When an electrical potential is applied, the axis of the peptide helices remains parallel to the membrane surface and does not reorient to give rise to a bundle of helix monomers that forms transmembrane channels via a 'barrel stave' mechanism. The penetration depth of alpha helices in relation to the position of phosphorus atoms in the unperturbed lipid leaflet is estimated at 3.2 A.     

Isolation and characterization of murine clonogenic osteoclast progenitors by cell surface phenotype analysis

MHC class I molecules bind short peptides for presentation to CD8+ T cells. The determination of the three-dimensional structure of various MHC class I complexes has revealed that both ends of the peptide binding site are composed of polar residues conserved among all human and murine MHC class I sequences, which act to lock the ends of the peptide into the groove. In the rat, however, differences in these important residues occur, suggesting the possibility that certain rat MHC class I molecules may be able to bind and present longer peptides. Here we have studied the peptide length preferences of two rat MHC class Ia molecules expressed in the TAP2-deficient mouse cell line RMA-S: RT1-A1c, which carries unusual key residues at both ends of the groove, and RT1.Aa which carries the canonical residues. Temperature-dependent peptide stabilization assays were performed using synthetic random peptide libraries of different lengths (7-15 amino acids) and successful stabilization was determined by FACS analysis. Results for two naturally expressed mouse MHC class I molecules revealed different length preferences (H2-Kb, 8-13-mer and H2-Db, 9-15-mer peptides). The rat MHC class Ia molecule, RT1-Aa, revealed a preference for 9-15-mer peptides, whereas RT1-A1c showed a more stringent preference for 9-12-mer peptides, thereby ruling out the hypothesis that unusual residues in rat MHC molecules allow binding of longer peptides.     

Structure, synthesis, and activity of dermaseptin b, a novel vertebrate defensive peptide from frog skin: relationship with adenoregulin

A novel antimicrobial peptide, designated dermaseptin b, was isolated from the skin of the arboreal frog Phyllomedusa bicolor. This 27-residue peptide amide is basic, containing 3 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. In helix-inducing solvent, dermaseptin b adopts an amphipathic alpha-helical conformation that most closely resembles class L amphipathic helixes, with all lysine residues on the polar face of the helix. The peptide exhibits growth inhibition activity in vitro against a broad spectrum of pathogenic microorganisms including yeast and bacteria as well as various filamentous fungi that are responsible for severe opportunistic infections accompanying acquired immunodeficiency syndrome and the use of immunosuppressive agents. Maximized pairwise sequence alignment of dermaseptin b and dermaseptin s, a 34-residue antimicrobial peptide previously isolated from Phyllomedusa sauvagii, reveals 81% amino acid identity. No other significant similarity was found between dermaseptin b and any prokaryotic or eukaryotic protein, but similarity was found with adenoregulin (38% amino acid postional identity), a 33-residue peptide that enhances binding of agonists to the A1 adenosine receptor. The synthetic replicates of dermaseptin b and adenoregulin displayed similar but nonidentical spectra of antimicrobial activity, and both peptides were devoid of lytic effect on mammalian cells. Accordingly, the observation that adenoregulin enhances binding of agonists to the adenosine receptor may in fact be a consequence of its ability to alter the structure of biological membranes and to produce signal transduction via interactions with the lipid bilayer, bypassing cell surface receptor interactions.     

Interaction of class A amphipathic helical peptides with phospholipid unilamellar vesicles

The exchangeable apolipoproteins are important in determining the structure/function properties of lipoproteins. These proteins typically contain varying amounts of amphipathic helices. Five model peptides, 18A, Ac-18A-NH2, Ac-18R-NH2, 37pA, and 37aA, have been designed to investigate variations of the amphipathic alpha-helix structural motif on their lipid-binding properties. These include the 18-residue peptides, 18A and Ac-18A-NH2, examples of class A helices, and Ac-18R-NH2, which has the positions of acidic and basic residues interchanged relative to 18A. Three larger peptides were also studied: 36A, a dimer of 18A, 37pA and 37aA, dimers of 18A coupled by Pro (18A-Pro-18A) and Ala (18A-Ala-18A), respectively. We report here the results of a thermodynamic characterization of the binding properties of these peptides to small unilamellar vesicles of POPC. Partition coefficients, Kp, were determined by fluorescence spectroscopy and binding enthalpies, deltaH, by titration calorimetry. These parameters were used to obtain the free energies, deltaG0, and entropies, deltaS0, of binding. The results of this study indicate Kp values on the order of 10(5), with interactions being enthalpically but not entropically favored in all cases. The presence of positively charged residues at the interface (18A and Ac-18A-NH2) enhances binding but has little effect on the extent of bilayer penetration. The presence of tandem repeats decreases lipid affinities for these small, highly curved bilayers. Our results are consistent with the idea that interaction appears to be confined largely to the surface, with some degree of penetration of the hydrophobic face of the helix into the interior of the bilayer.     

How cytidylyltransferase uses an amphipathic helix to sense membrane phospholipid composition

CT responds to properties of PC-depleted membranes: increased negative charge density, which concentrates the enzyme at the membrane surface, and lipid packing perturbations, which create holes in the membrane surface into which the hydrophobic side chains of the amphipathic helix of domain M can intercalate. The PC-deficient lipid surface appears capable of catalysing the folding of domain M into an alpha-helix. The determinants on domain M which create a preference for anionic lipids are: (i) strips of interfacial lysines; (ii) three serines within the non-polar face; (iii) three interfacial glutamates whose protonation state appears to be sensitive to the surface charge. Phosphorylation of the domain adjacent to domain M decreases the membrane affinity of the amphipathic helix, perhaps by an ion-pairing competition. The mechanism whereby the stabilization of an alpha-helical conformation of domain M is transduced into a conformational change in the catalytic domain is the key question for future exploration.     

Relationship of sidechain hydrophobicity and alpha-helical propensity on the stability of the single-stranded amphipathic alpha-helix

The aim of the present investigation is to determine the effect of alpha-helical propensity and sidechain hydrophobicity on the stability of amphipathic alpha-helices. Accordingly, a series of 18-residue amphipathic alpha-helical peptides has been synthesized as a model system where all 20 amino acid residues were substituted on the hydrophobic face of the amphipathic alpha-helix. In these experiments, all three parameters (sidechain hydrophobicity, alpha-helical propensity and helix stability) were measured on the same set of peptide analogues. For these peptide analogues that differ by only one amino acid residue, there was a 0.96 kcal/mole difference in alpha-helical propensity between the most (Ala) and the least (Gly) alpha-helical analogue, a 12.1-minute difference between the most (Phe) and the least (Asp) retentive analogue on the reversed-phase column, and a 32.3 degrees C difference in melting temperatures between the most (Leu) and the least (Asp) stable analogue. The results show that the hydrophobicity and alpha-helical propensity of an amino acid sidechain are not correlated with each other, but each contributes to the stability of the amphipathic alpha-helix. More importantly, the combined effects of alpha-helical propensity and sidechain hydrophobicity at a ratio of about 2:1 had optimal correlation with alpha-helix stability. These results suggest that both alpha-helical propensity and sidechain hydrophobicity should be taken into consideration in the design of alpha-helical proteins with the desired stability.     

Lipid-induced organization of a primary amphipathic peptide: a coupled AFM-monolayer study

To better understand the nature of the mechanism involved in the membrane uptake of a vector peptide, the interactions between dioleoylphosphatidylcholine and a primary amphipathic peptide containing a signal peptide associated with a nuclear localization sequence have been studied by isotherms analysis of mixed monolayers spread at the air-water interface. The peptide and the lipid interact through strong hydrophobic interactions with expansion of the mean molecular area that resulted from a lipid-induced modification of the organization of the peptide at the interface. In addition, a phase separation occurs for peptide molar fraction ranging from about 0.08 to 0.4 Atomic force microscopy observations made on transferred monolayers confirm the existence of phase separation and further reveal that mixed lipid-peptide particles are formed, the size and shape of which depend on the peptide molar fraction. At low peptide contents, round-shaped particles are observed and an increase of the peptide amount, simultaneously to the lipidic phase separation, induces morphological changes from bowls to filamentous particles. Fourier transform infrared spectra (FTIR) obtained on transferred monolayers indicate that the peptide adopts a beta-like structure for high peptide molar fractions. Such an approach involving complementary methods allows us to conclude that the lipid and the peptide have a nonideal miscibility and form mixed particles which phase separate.     

Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin

Membranes have a potent ability to promote secondary structure formation in a wide range of membrane-active peptides, believed to be due to a reduction through hydrogen bonding of the energetic cost of partitioning peptide bonds. This process is of fundamental importance for understanding the mechanism of action of toxins and antimicrobial peptides and the stability of membrane proteins. A classic example of membrane-induced folding is the bee-venom peptide melittin that is largely unstructured when free in solution, but strongly adopts an amphipathic alpha-helical conformation when partitioned into membranes. We have determined the energetics of melittin helix formation through measurements of the partitioning free energies and the helicities of native melittin and of a diastereomeric analog with four d-amino acids (d4,l-melittin). Because D4,l-melittin has little secondary structure in either the free or bound forms, it serves as a model for the experimentally inaccessible unfolded bound form of native melittin. The partitioning of native melittin into large unilamellar phosphocholine vesicles is 5.0(+/-0.7) kcal mol-1 more favorable than the partitioning of d4,l-melittin (1 cal=4.186 J). Differences in the circular dichroism spectra of the two forms of melittin indicate that bound native melittin is more helical than bound d4, l-melittin by about 12 residues. These findings disclose that the free energy reduction per residue accompanying the folding of melittin in membrane interfaces is about 0.4 kcal mol-1, consistent with the hypothesis that hydrogen bonding reduces the high cost of partitioning peptide bonds. A value of 0.6 kcal mol-1 per residue has been observed for beta-sheet formation by a hexapeptide model system. These two values provide a useful rule of thumb for estimating the energetic consequences of membrane-induced secondary structure formation.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic alpha-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no alpha-helicity in buffer or liposomes, exhibits a modest degree of alpha-helical structure in the presence of the alpha-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an alpha-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata

BACKGROUND: Killer lymphocytes secrete perforin, a 67 kDa protein that initiates T-cell cytolysis following aggregation and pore formation in target membranes. The resulting pores cause a breakdown of the transmembrane osmotic gradient and allow other cytolytic mediators to enter the target cell and initiate apoptosis. The cytolytic domain resides within the first 34 residues of the amino terminus of perforin, with residues 1-19 being sufficient for cytolytic activity. RESULTS: The solution structure of a 22-residue synthetic peptide (P22), corresponding to the amino terminus of human perforin, has been determined using high resolution nuclear magnetic resonance spectroscopy in the presence and absence of perdeuterated detergent (SDS) micelles. In aqueous solution, P22 exists mainly in a random conformation. However, it adopts a hook-like structure at the carboxyl terminus in the presence of SDS micelles when the positively charged residues cluster to form a turn that provides a binding surface to the negatively charged sulfate headgroups. CONCLUSIONS: The strong electrostatic interaction between the cationic region of the P22 peptide and the lipid headgroups probably weakens the membrane, facilitating insertion of the relatively neutral/hydrophobic stretch of P22, and is representative of the initial step of the lytic pathway. The structural model described here is probably relevant to understanding the mechanisms of other cationic antimicrobial peptides.     

Simulation of voltage-dependent interactions of alpha-helical peptides with lipid bilayers

Pore formation in lipid bilayers by channel-forming peptides and toxins is thought to follow voltage-dependent insertion of amphipathic alpha-helices into lipid bilayers. We have developed an approximate potential for use within the CHARMm molecular mechanics program which enables one to simulate voltage-dependent interaction of such helices with a lipid bilayer. Two classes of helical peptides which interact with lipid bilayers have been studied: (a) delta-toxin, a 26 residue channel-forming peptide from Staphylococcus aureus; and (b) synthetic peptides corresponding to the alpha 5 and alpha 7 helices of the pore-forming domain of Bacillus thuringiensis CryIIIA delta-endotoxin. Analysis of delta-toxin molecular dynamics (MD) simulations suggested that the presence of a transbilayer voltage stabilized the inserted location of delta-toxin helices, but did not cause insertion per se. A series of simulations for the alpha 5 and alpha 7 peptides revealed dynamic switching of the alpha 5 helix between a membrane-associated and a membrane-inserted state in response to a transbilayer voltage. In contrast the alpha 7 helix did not exhibit such switching but instead retained a membrane associated state. These results are in agreement with recent experimental studies of the interactions of synthetic alpha 5 and alpha 7 peptides with lipid bilayers.     

Structure effects of double D-amino acid replacements: a nuclear magnetic resonance and circular dichroism study using amphipathic model helices

D-Amino acid replacements and the determination of resulting structural changes are a useful tool to recognize amphipathic helices in biologically active peptides such as neuropeptide Y and corticotropin-releasing factor. In this paper the secondary structures of one amphipathic alpha-helical peptide and its double D-amino acid analog have been determined by means of 1H NMR and CD spectroscopies under equivalent conditions. The chemical shifts (NH and C alpha H) and the analysis of nuclear Overhauser effects show a split of the continuous helix for the all-L peptide into two helices at the position of double D-amino acid replacement. Hydrogen exchange rates correlate with water accessibilities in the hydrophobic/hydrophilic face and confirm the amphipathic helical structure in the all-L peptide as well as in its double D-amino acid analog. A significantly accelerated hydrogen isotope exchange rate is observed for the D-Ala9 backbone proton, implying an increased flexibility at that position. These results show that the incorporation of an adjacent pair of D-amino acids only causes a local change in structure and flexibility, which makes the double D replacement interesting as a tool for specific helix-disturbing modifications to search for helical conformations in biologically active peptides.     

Membrane fusion induced by 11-mer anionic and cationic peptides: a structure-function study

We recently demonstrated that an amphipathic net-negatively charged peptide consisting of 11 amino acids (WAE 11) strongly promotes fusion of large unilamellar liposomes (LUV) when anchored to a liposomal membrane [Pecheur, E. I., Hoekstra, D., Sainte-Marie, J., Maurin, L., Bienvenue, A., and Philippot, J. R. (1997) Biochemistry 36, 3773-3781]. To elucidate a potential relationship between peptide structure and its fusogenic properties and to test the hypothesis that specific structural motifs are a prerequisite for WAE-induced fusion, three 11-mer WAE-peptide analogues (WAK, WAEPro, and WAS) were synthesized and investigated for their structure and fusion activity. Structural analysis of the synthetic peptides by infrared attenuated total reflection spectroscopy reveals a distinct propensity of each peptide toward a helical structure after their anchorage to a liposomal surface, emphasizing the importance of anchorage on conveying a secondary structure, thereby conferring fusogenicity to these peptides. However, whereas WAE and WAK peptides displayed an essentially nonleaky fusion process, WAS- and WAEPro-induced fusion was accompanied by substantial leakage. It appears that peptide helicity as such is not a sufficient condition to convey optimal fusion properties to these 11-mer peptides. Studies of changes in the intrinsic Trp fluorescence and iodide quenching experiments were carried out and revealed the absence of migration of the Trp residue of WAS and WAEPro to a hydrophobic environment, upon their interaction with the target membranes. These results do not support the penetration of both peptides as their mode of membrane interaction and destabilization but rather suggest their folding along the vesicle surface, posing them as surface-seeking helixes. This is in striking contrast to the behavior observed for WAE and WAK, for which at least partial penetration of the Trp residue was demonstrated. These results indicate that subtle differences in the primary sequence of a fusogenic peptide could induce dramatic changes in the way the peptide interacts with a bilayer, culminating in equally drastic changes in their functional properties. The data also reveal a certain degree of sequence specificity in WAE-induced fusion.     

Folding of the presequence of yeast pAPI into an amphipathic helix determines transport of the protein from the cytosol to the vacuole

To investigate the role of the 17 residues long presequence (p17) in the transport of the precursor of yeast API (pAPI) from the cytosol to the vacuole we have studied the effects of point mutations upon its conformation and on the process of transport. 1H NMR analysis of p17 indicates that in aqueous solution 26% of the molecules have the 4-12 segment folded into an helix. The hydrophobic environment provided by SDS micelles promotes the folding of 54% of the p17 molecules into a 5-16 amphipathic alpha-helix. Both Schiffer-Edmunson helical wheel analysis of segment 4-12 and residue hydrophobic moments calculated considering all possible side-chain orientations between 80 and 120 degrees, indicate the amphipathic character of the helixes assembled in water and detergent. Charge interactions between the dipole pairs N-Glu2Glu3 and C-Lys12Lys13 are essential for helix stability and condition pAPI transport. Substitution of either Pro2Pro3 or Lys2Lys3 for Glu2Glu3, results in moderate destabilization of the helix, decreases protein targeting to the vacuolar membrane and partly inhibits translocation of the protein to the vacuolar lumen. Replacement of either Pro12Pro13 or Glu12Glu13 for Lys12Lys13, causes a major disruption of the helix, decreases protein targeting and blocks completely the translocation of the protein to the vacuolar lumen. Replacement of Gly7 for Ile7, a substitution which is known to destabilize alpha-helixes in peptides and proteins as a result of the peptide bond to the solvent at Gly residues, produces similar effects as the substitutions for the K12K13 pair. The effects of Gly7 on helix stability and protein transport are partly reversed by introduction of Asp residues at positions 2 and 3 and Ala at position 4. Replacements such as Arg2 for Glu2, or Arg6 for Glu6, which change the net and local charges of the presequence without altering its conformation, have no effect on the protein transport. These results provide direct evidence of the involvement of the presequence in the transport of pAPI from the cytosol to the vacuole. They show that folding of the pAPI presequence is conditioned by the physical/chemical properties of the environment and is critical for targeting the protein to the vacuolar membrane and for its translocation to the vacuolar lumen.