Amphipathic Alpha-helical Peptides Research Articles

Mode of action of linear amphipathic alpha-helical antimicrobial peptides.

The increasing resistance of bacteria to conventional antibiotics resulted in a strong effort to develop antimicrobial compounds with new mechanisms of action. Antimicrobial peptides seem to be a promising solution to this problem. Many studies aimed at understanding their mode of action were described in the past few years. The most studied group includes the linear, mostly alpha-helical peptides. Although the exact mechanism by which they kill bacteria is not clearly understood, it has been shown that peptide-lipid interactions leading to membrane permeation play a role in their activity. Membrane permeation by amphipathic alpha-helical peptides can proceed via either one of the two mechanisms: (a) transmembrane pore formation via a "barrel-stave" mechanism; and (b) membrane destruction/solubilization via a "carpet-like" mechanism. The purpose of this review is to summarize recent studies aimed at understanding the mode of action of linear alpha-helical antimicrobial peptides. This review, which is focused on magainins, cecropins, and dermaseptins as representatives of the amphipathic alpha-helical antimicrobial peptides, supports the carpet-like rather the barrel-stave mechanism. That these peptides vary with regard to their length, amino acid composition, and next positive charge, but act via a common mechanism, may imply that other linear antimicrobial peptides that share the same properties also share the same mechanism.

Influence of the angle subtended by the positively charged helix face on the membrane activity of amphipathic, antibacterial peptides.

To investigate the influence of the angle subtended by the positively charged helix face on membrane activity, six amphipathic alpha-helical peptides with angles between 80 degrees and 180 degrees, but with retained hydrophobicity, hydrophobic moment, and positive overall charge, were designed starting from the sequence of the antibacterial peptide magainin 2. CD investigations revealed that all analogs are in an alpha-helical conformation in vesicle suspension. The ability of the peptides to induce dye release from negatively charged phosphatidylglycerol (PG) vesicles decreased with increasing angle. However, peptides with a large angle of positively charged residues (140-180 degrees) exhibited a considerably higher permeabilizing activity at zwitterionic phosphatidylcholine (PC) and mixed PC/PG (3:1) vesicles than analogs with a small angle (80-120 degrees). In addition, analogs with large angles were more active in antibacterial and hemolytic assays. The antibacterial specificity of these analogs was decreased. Binding investigations showed that peptide binding is favored by a large angle and a high content of negatively charged phospholipid. In contrast, a small angle and a low negative membrane charge enhanced the membrane-permeabilizing efficiency of the bound peptide fraction. All analogs stabilized the bilayer phase of phosphatidylethanolamine over the inverted hexagonal phase. Therefore, a class L mechanism of permeabilization can be excluded. Furthermore, the analogs do not act by the induction of positive curvature strain or by a "carpet-like" mechanism. Our results are in accordance with a pore mechanism: The membrane-permeabilizing efficiency of analogs with enhanced angle of positively charged residues is reduced due to electrostatic repulsion between adjacent helices within the pore, thus resulting in a decreased pore-forming probability and/or pore destabilization.

RS-66271, a C-terminally substituted analog of human parathyroid hormone-related protein (1-34), increases trabecular and cortical bone in ovariectomized, osteopenic rats.

It was predicted from the amino acid sequence of the bone anabolic peptides, parathyroid hormone (PTH) (1-34) and PTH related protein (PTHrP) (1-34), that the C-terminal amino acids form an amphipathic alpha-helix. Therefore, we substituted a model amphipathic alpha-helical peptide (MAP) sequence in the C-terminal region of hPTHrP(1-34), obtaining RS-66271 ([MAP1-10]22-31 hPTHrP(1-34)-NH2). The anabolic activities of RS-66271 and hPTHrP(1-34) were evaluated in 3-month-old, ovariectomized (OVX) osteopenic rats. Subcutaneous injection of hPTHrP(1-34) at 80 micrograms/kg/day partially reversed estrogen depletion trabecular bone loss but was ineffective in the cortex. In contrast, RS-66271 dose-relatedly reversed loss at both sites and, at 80 micrograms/kg/day, returned both trabecular and cortical bone calcium to the level of sham-operated controls. Histomorphometric analysis showed significantly elevated bone formation rates over vehicle-treated OVX in both trabecular and cortical tibial bone following treatment with RS-66271. Electron microscopy showed an increase in the relative surface area of vertebral trabeculae covered by osteoblasts in animals treated with RS-66271. These studies demonstrate that the C-terminal amino acids of hPTHrP(1-34) can be replaced by a model amphipathic helix and that the new chemical entity has greater anabolic activity than the parent peptide. The results suggest that RS-66271 may be a candidate molecule for the treatment of human osteoporosis.

Hydrophobic effects on antibacterial and channel-forming properties of cecropin A-melittin hybrids.

The design of cecropin-melittin hybrid analogues is of interest due to the similarities in the structure of the antimicrobial peptides cecropin and melittin but differences in their lytic properties. We suspected that a hydrophobic residue in position 2 of milittin (Ile8 in the hybrid) plays an important role in the activity of the 15-residue hybrid, KWKLFKKIGAVLKVL-NH2, [CA(1-7)M(2-9)NH2] and have now examined its role in the analogue toward five test bacteria. Deletion of Ile8 reduced activity, and it was not restored by lengthening to 15 residues by addition of another threonine at the C-terminus. Replacement of Ile8 by a hydrophobic leucine maintained good activity and Ala8 was equally active for four organisms, although less active against Staphylococcus aureus. Replacement by the hydrophilic Ser8 strongly reduced potency against all five organisms. Deletion of Leu15 decreased activity, but addition of Thr16 maintained good activity. The presence of hydrophobic residues appears to have a significant effect on the process of antibacterial activity. These peptide analogues showed voltage-dependent conductance changes and are capable of forming ion-pores in planar lipid bilayers. The antibacterial action of the peptides is thought to be first an ionic interaction with the anionic phosphate groups of the membrane followed by interaction with the hydrocarbon core of the membrane and subsequent reorientation into amphipathic alpha-helical peptides that form pores (ion-channels), which span the membrane. The analogue also showed an increase in alpha-helicity with an increase in hexafluoro 2-propanol concentration.

Structure Effects of Double D-Amino Acid Replacements: A Nuclear Magnetic Resonance and Circular Dichroism Study Using Amphipathic Model Helixes

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.

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.

Peptide binding domains determined through chemical modification of the side-chain functional groups

A clear understanding of the specific secondary structure and binding domain resulting from the interactions of proteins and peptides with lipid surfaces will provide insight into the specific functions of biologically active molecules. We have shown in earlier studies that the stationary phases used in reverse-phase high-performance liquid chromatography represent a model artificial lipid surface for the study of induced conformational states of peptides on lipid interaction. We have now used reverse-phase high-performance liquid chromatography to determine the binding domains of peptides and, by extension, of proteins to a lipid surface. This approach consists of performing chemical modifications of specific amino acid side-chain functionalities after the interaction of the peptides with the reverse-phase high-performance liquid chromatography C18 groups. The susceptibility to oxidation was also studied after binding of the same peptides to liposomes. Oxidation of a single methionine residue "walked" through an amphipathic alpha-helical 18-mer peptide was selected to illustrate this approach. The extent of oxidation was found to be clearly dictated by the accessibility of the methionine residue to the aqueous mobile phase. The binding domain found for the peptide in its lipid-induced conformational state was unequivocally the entire hydrophobic face of the amphipathic alpha-helix.

Lactam bridge stabilization of alpha-helical peptides: ring size, orientation and positional effects.

A series of 14 residue amphipathic alpha-helical peptides, in which the sidechains of glutamic acid and lysine have been covalently joined, was synthesized in order to determine the effect of spacing, position and orientation of these lactam bridges. It was found that although an (i, i+3) spacing would position the lactam bridge on the same face of the helix, these lactams with 18-member rings were actually helix-destabilizing regardless of position or location. On the other hand, (i, i+4) lactams with 21-member rings were helix-stabilizing but this was dependent on orientation. Glutamic acid-lysine lactams increased the helical content of the peptide when compared with their linear homologue in benign conditions (50 mM KH2PO4, 100 mM KCl, pH 7). Two Glu-Lys (i, i+4) lactams located at the N- and C-termini gave rise to a peptide with greater than 99% helical content in benign conditions. Peptides with Lys-Glu oriented lactams were random structures in benign conditions but in the presence of 50% TFE could be induced into a helical conformation. The stability of the single-stranded alpha-helices, as measured by thermal denaturations in 25% TFE indicated that Glu-Lys oriented lactam bridges stabilized the helical conformation relative to the linear unbridged peptide. One Glu-Lys lactam in the middle of the peptide was more effective at stabilizing helical structure than two Glu-Lys lactams positioned one at each end of the molecule. The lactams with the Lys-Glu orientation were destabilizing relative to the unbridged peptide. This study demonstrates that correct orientation and position of a lactam bridge is critical in order to design peptides with high helical content in aqueous media.

Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils.

A series of polypeptides containing 9, 12, 16, 19, 23, 26, 30, 33, and 35 amino acid residues was designed to investigate the effects of peptide chain length on the formation and stability of two-stranded alpha-helical dimers or coiled coils. These peptides were synthesized by the solid-phase method, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and characterized by RP-HPLC, amino acid composition analysis, and mass spectrometry. The amphipathic alpha-helical peptides were designed to dimerize by interchain hydrophobic interactions at positions a and d and interchain salt bridges between lysine and glutamic acid residues at positions e and g of the repeating heptad sequence of Glu-Ile-Glu-Ala-Leu-Lys-Ala (g-a-b-c-d-e-f). The ability of these peptides to form alpha-helical structures in the presence and absence of a helix-inducing reagent (trifluoroethanol) was monitored by circular dichroism spectroscopy. The helicity of the peptides increased with increasing chain length in a cooperative manner. A minimum of three heptads corresponding to six helical turns was required for a peptide to adopt the two-stranded alpha-helical coiled coil conformation in aqueous medium. The increased stability of the peptides as a result of an increase in hydrophobic interactions (chain length) was demonstrated by the shift in the transitions of the guanidine hydrochloride (Gdn.HCl) denaturation and thermal unfolding profiles. The concentrations of denaturant (Gdn.HCl) required to achieve 50% denaturation are 3.2, 4.9, 6.9, and 7.5 M for peptides 23r, 26r, 30r, and 33r, respectively, in aqueous medium. However, the effect of a chain length increase on coiled-coil stability was not additive. The melting temperature, Tm, at which 50% of the helicity is lost, increased by 34 degrees C in changing the peptide chain length from 23 to 26; however, that shift was only 14 degrees C when the chain length was increased from 30 to 33 residues. These results are consistent with a chain length dependent cooperative folding of the peptides into coiled coils.

A single-stranded amphipathic .alpha.-helix in aqueous solution: Design, structural characterization, and its application for determining .alpha.-helical propensities of amino acids

In order to investigate the positional effect of alpha-helical propensities of amino acids in an amphipathic alpha-helix, an amphipathic alpha-helical model peptide (Ac-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys-Ala-Ala-Lys-Glu-Ala-Glu-Lys- amide) was designed and characterized by circular dichroism and 2D-NMR spectroscopies. This peptide contains 65% alpha-helical structure in solution, and its monomeric molecular weight in aqueous solution was determined by size-exclusion chromatography. The independence of alpha-helical structure and stability on peptide concentration demonstrates that helix formation of this peptide is a monomolecular process. To compare the effect of substitutions in the hydrophobic and hydrophilic face of the helix on structure and stability, a leucine, alanine, or glycine was individually substituted in the hydrophobic face (position 9) or hydrophilic face (position 7) of the model peptide. The change in helix content and stability upon substitution was measured by circular dichroism studies in the absence and presence of TFE or urea. The results indicate that each amino acid has a different helix propensity when it is located in the hydrophobic face versus hydrophilic face and the effect of substitution is more significant in the hydrophobic face. This single-stranded amphipathic alpha-helical peptide provides an appropriate model system to determine helix propensities of amino acids on both hydrophobic and hydrophilic faces.

Promotion of β-structure by interaction of diabetes associated polypeptide (amylin) with phosphatidylcholine

The interaction of the diabetes associated polypeptide (amylin) with dimyristoylphosphatidylcholine (DMPC) was assessed by measurements of turbidity (absorbance at 400 nm) and secondary structure by CD spectroscopy. In trifluoroethanol, human amylin adopts a highly alpha-helical conformation while the rat peptide is less structured. In water, the rat peptide is largely disordered and the human peptide exhibits a combination of alpha- and beta-structures. Mixtures of DMPC and the rat peptide have no effect on either the turbidity of the DMPC or the CD spectrum of the peptide. By contrast, mixtures of the human peptide with DMPC form relatively clear mixtures similar to those observed with amphipathic alpha-helical peptides, but the structure adopted, based on the CD spectrum, is largely beta. These data demonstrate that fundamental differences in the structures adopted by amylins from human and rat species exist in mixtures with DMPC and suggest that these differences may be related to the formation of amyloid fibrils in the human amylin peptide which are not observed in the rat peptide.

Nuclear magnetic resonance studies and molecular dynamics simulations of the solution conformation of a 'designed', alpha-helical peptide.

The complete three-dimensional structure in methanol of an amphipathic alpha-helical peptide, that has been designed by taking into account the three-dimensional structures of small haemolytic peptides, secondary structure prediction algorithms and the well documented literature on alpha-helix stabilizing factors, has been elucidated by two-dimensional NMR spectroscopy. Initially various two-dimensional spectra (COSY, TOCSY, and NOESY) allowed the complete sequence specific assignment of all signals in the 1H spectrum. Consequently trial structures were generated which were then subjected to molecular dynamics simulations using 121 NOE-derived distances and 25 vicinal coupling constant values as structural restraints to give a final set of calculated structures. These structures are in complete agreement with the results of a circular dichroism study and reveal that the peptide adopted a highly ordered alpha-helical conformation. Details of the structure which throw light on future peptide/protein design are discussed.

Interaction with phospholipid bilayers, ion channel formation, and antimicrobial activity of basic amphipathic alpha-helical model peptides of various chain lengths.

Basic amphipathic alpha-helical peptides Ac-(Leu-Ala-Arg-Leu)3 or 4-NHCH3 (4(3) or 4(4)) and H-(Leu-Ala-Arg-Leu)3-(Leu-Arg-Ala-Leu)2 or 3-OH (4(5) or 4(6)) were synthesized and studied in terms of their interactions with phospholipid membranes, biological activity, and ion channel-forming ability. CD study of the peptides showed that they form alpha-helical structures in the presence of phospholipid liposomes and thus they have amphipathic distribution of the side chains along the axis of the helix. A leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides possess a highly potent ability to perturb the membrane structure. Membrane current measurements using the planar lipid bilayer technique revealed that the peptide 4(6), which was long enough to span the lipid bilayer in the alpha-helical structure, formed cation-selective ion channels at a concentration of 0.5 microM in a planar diphytanoylphosphatidylcholine bilayer. In contrast, other shorter peptides failed to form discrete and stable channels though they occasionally induced an increase in the membrane current with erratic conductance levels. The probability of detecting a conductance increase was in the order of 4(6) greater than 4(5) greater than 4(4) greater than 4(3), which corresponds to the order of the peptide chain lengths. Furthermore, 4(6) but not 4(5) showed an antimicrobial activity against both Gram-positive and -negative bacteria. The structure of ion channels formed by 4(6) and the relationship between the peptide chain length and biological activity of the synthetic peptides are discussed.

Ion channels formed by a highly charged peptide

A peptide (MA-beta) corresponding to a segment of the nicotinic acetylcholine receptor (AChR) that has amphipathic alpha-helical periodicity forms ion channels in artificial phospholipid bilayers. The MA-beta ion channels are very stable, comprise two discrete conductance states, and undergo rapid, flickering-type closings. The discrete-conductance ion channels formed by MA-beta contrast with the continuous-conductance ion channels formed by a peptide (M2-delta) identical in sequence with M2 [Oiki, S., Danho, W., Madison, V., & Montal, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8703-8707], a putative transmembrane segment of the AChR. Neither MA-beta nor M2-delta sufficiently mimics the electrophysiological properties of the native AChR. We suggest that peptide ion channels can be classified into at least three general groups: discrete-conductance channels, such as MA-beta; continuous-conductance channels, such as M2-delta; and membrane disruptors, such as those formed by short, amphipathic alpha-helical peptides.

Minimal peptide length for interaction of amphipathic .alpha.-helical peptides with phosphatidylcholine liposomes

The interactions of a series of amphipathic alpha-helical peptides containing from 6 to 18 amino acid residues with dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) were studied by optical and calorimetric methods. Several peptides rapidly decreased the turbidity of DMPC and DPPC liposomes when mixed at the phase transition temperatures of the lipids. The extent of the clearing depended upon the chain length of the peptides, with the most effective clearing attained with peptides 10-12 residues in length. An eight-residue peptide was somewhat less effective and a six-residue peptide had no effect on liposome structure. The peptides formed small micellar structures, as judged by gel filtration chromatography. The effects of the peptides on the phase transitions of the lipids were examined by differential scanning calorimetry. The peptides that were most effective in disrupting the liposomes and forming clear micelles were also most effective in reducing the enthalpy of the gel to liquid-crystalline phase transition of the lipid. The addition of DMPC or DPPC liposomes to the peptides increased the magnitude of the negative bonds at 208 and 222 nm in circular dichroism measurements, consistent with the expected formation of alpha-helical structure on binding to lipid. The extent of burial of the single tryptophan residue in the peptides was determined by fluorescence spectroscopy. In peptides that bound to lipid, the tryptophan was in a less solvent-exposed environment in the presence of lipid, as evidenced by a blue shift in the fluorescence emission maximum of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of peptide hormones of the amphipathic helix class using the helical hydrophobic moment algorithm.

Eisenberg's helical hydrophobic moment (less than mu H greater than) algorithm was applied to the analysis of the primary structure of amphipathic alpha-helical peptide hormones and an optimal method for identifying other peptides of this class determined. We quantitate and compare known amphipathic helical peptide hormones with a second group of peptides with proven nonamphipathic properties and determine the best method of distinguishing between them. The respective means of the maximum 11 residue less than mu H greater than for the amphipathic helical and control peptides were 0.46 (+/-/-0.07) and 0.33 (0.07) (P + 0.004). To better reflect the amphipathic potential of the entire peptide, the percent of 11 residue segments in each peptide above a particular less than mu H greater than was plotted vs less than mu H greater than. The resulting curves are referred to as HM-C. The mean HM-C (of the two groups) was highly significantly different such that the HM-C method was superior to others in its ability to distinguish amphipathic from nonamphipathic peptides. Several potential new members of this structural class were identified using this approach. Molecular modeling of a portion of one of these, prolactin inhibitory factor, reveals a strongly amphipathic alpha helix at residues 4-21. This computer-based method may enable rapid identification of peptides of the amphipathic alpha-helix class.