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The high costs of artificial pulmonary surfactants, ranging in hundreds per kilogram of body weight, used for treating the respiratory distress syndrome (RDS) premature babies have limited their applications. We have extensively studied soy lecithins and higher alcohols as lipid alternatives to expensive phospholipids such as DPPC and PG. As a substitute for the proteins, we have synthesized the peptide Hel 13-5D3 by introducing D-amino acids into a highly lipid-soluble, basic amphiphilic peptide, Hel 13-5, composed of 18 amino acid residues. Analysis of the surfactant activities of lipid-amphiphilic artificial peptide mixtures using lung-irrigated rat models revealed that a mixture (Murosurf SLPD3) of dehydrogenated soy lecithin, fractionated soy lecithin, palmitic acid (PA), and peptide Hel 13-5D3 (40:40:17.5:2.5, by weight) superior pulmonary surfactant activity than a commercially available pulmonary surfactant (beractant, Surfacten®). Experiments using ovalbumin-sensitized model animals revealed that the lipid-amphiphilic artificial peptide mixtures provided significant control over an increase in the pulmonary resistance induced by premature allergy reaction and reduced the number of acidocytes and neutrophils in lung-irrigated solution. The newly developed low-cost pulmonary surfactant system may be used for treatment of a wide variety of respiratory diseases.

In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air-water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500-1700cm(-1)) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the alpha-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the alpha-helix to the extended beta-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the beta-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air-water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface.

In order to investigate the influence of the alanine residue at position 1 of AM-toxin I (cyclic tetradepsipeptide) on necrotic activity for apple leaves, three analogs, [l-2-aminobutanoic acid1] AM-toxin I, [l-leucine1] AM-toxin I and [l-phenylalanine1] AM-toxin I, were synthesized by the conventional method for peptide synthesis. Spectra of 1H-n.m.r., u.v. and CD of the analogs were similar to those of natural AM-toxin I. The analogs showed appreciable biological activity including host-specificity, indicating that considerable variation in the size of side chain at position 1 is allowed for the induction of necrotic activity.

Recent investigation have indicated that cyclic dodeca- and tetradecapeptides, cyclo(-Leu-Orn-Leu-Orn-D-Phe-Pro)2 (Orn-DLL-12) and cyclo(-Leu-Orn-Leu-Orn-Leu-D-Phe-Pro)2 (Orn-DLL-14), which are designed on the basis of a cyclic beta-structural antibiotic, gramicidin S (GS), inhibit the growth of Gram-positive and -negative bacteria with high potency [Ando, S., Nishikawa, H., Takiguchi, H., Lee, S. & Sugihara, G. (1993) Biochim. Biophys. Acta 1147, 42-49]. In this study we designed and synthesized two analogs, Lys-DLL-12 and Lys-DLL-14, in which four Orn residues in Orn-DLL-12 and Orn-DLL-14 were replaced by Lys residues, respectively, and investigated their interactions with model membranes in terms of CD and dye-leakage experiments, antimicrobial activity and lytic activity for human erythrocytes. Both peptides newly designed showed no antimicrobial activity and no lytic activity of erythrocytes. The present CD study showed that the presence of neutral liposomes and of acidic liposomes of natural or synthetic phospholipids results in no remarkable conformational difference between Orn-DLL-12/-14. The leakage experiment showed a clear relation between the antimicrobial activity and the leakage ability in acidic synthetic phospholipid liposomes but no correlation in acidic natural ones. The difference in hydrophobic and hydrophilic balance between Orn-DLL-12/14 and Lys-DLL-12/14 (derived from the increasing hydrophobicity due to an increase of four methylene units by the substitution of Lys for Orn) may be one of the important factors in the drastic decrease in activity.

A useful set of empirical rules is put forward to predict the conformations of cyclic tetrapeptides and cyclic tetradepsipeptides on the basis of primary structure, briefly presented as follows: 1. A conformation allowing an intramolecular hydrogen bond (IMHB) of γ-turn is preferred, and an ester bond always adopts a trans form. 2. On a right-handed peptide ring, the carbonyl group acylating a D residue is oriented to the upper side of the main ring. 3. The carbonyl group acylating a d proline or an N-methyl-d-amino acid residue is oriented to the lower side of the ring, forming a cis bond. 4. The lddl configurational sequence adopts a cis-trans-cis-trans backbone with Ci symmetry. 5. A glycine residue behaves as a d residue in an l-peptide. Conformations of cyclotetrapeptides containing two glycine residues at diametric positions or containing an N-methyl-dehydroamino acid residue are predicted by use of appendices of rule 5. Almost all conformations of cyclic tetrapeptides are predicted by these rules. Energetical rationalization of the rules and prediction of possible new conformations are described. Conformations of cyclo (-l-Pro-l-Leu-d-Tyr(Me)-l-Ile-)(1) and cyclo (-l-Pro-d-Leu-d-Tyr(Me)-l-Ile)(2) are compared. Results of n.m.r. experiments showed that compound 1 adopts a unique cis-trans-trans-trans backbone with a γ-turn IMHB, and 2 has a cis-trans-cis-trans backbone with Ci symmetry. These observations confirmed the rules described above. Peptides 1 and 2 are the first diastereomeric peptides with trans (ld) and cis (dd) secondary amide bonds.

The novel fluorescent amino acid, L-1-pyrenylalanine (L-Pya), was prepared by the asymmetric hydrogenation of cyclic dehydrodipeptide. Fluorescent enkephalins containing one or two Pya residues at position 1,4 or 5 of [D-Ala2, Leu5]enkephalin were synthesized by the solution method. Mono-Pya-enkephalins showed strong fluorescence intensities and potent binding affinities with specificity and selectivity for opiate receptors. However, di-Pya-enkephalins showed markedly decreased receptor binding affinities. These results indicate that the incorporation of two Pya residues into enkephalin makes the peptide unable to interact with the opiate receptors, although introduction of one Pya residue is effective to elicit a specific receptor interaction. Di-Pya-enkephalins showed intramolecular excimer spectra, indicating that the peptides are able to take possible folded conformations.

The effect of ester bond on the conformation of peptide molecule was studied by designing and synthesizing a model tetradepsipeptide cyclo(-L-Ala-L-Hmb-)2 and by analyzing the conformation both theoretically and experimentally. Theoretical analysis showed that both ester and peptide bonds in the calculated low-energy conformations within 3 kcal/mol of the global minimum take a trans but distorted configuration. The distortion is larger in ester bonds than in peptide bonds. Further, the four carbonyls project from one side of the plane of the cyclic backbone, whereas the side chains project from the other side. These results are consistent with the experimental results obtained by NMR measurement; first, the coupling constant deduced from 1H-NMR species in DMSO-d6 is consistent with the dihedral angles of the calculated low-energy conformations; second, results of NOE measurement can reproduce the calculated configuration of the carbonyls and side chains. From the consistency between theoretical and experimental results, it is concluded that this model tetradepsipeptide takes an all-trans backbone conformation in solution and this backbone conformation is stabilized by large distortion in the ester bond, which compensates the strain resulted from the 12-membered cyclic backbone structure consisting only of L-residues.

A number of cyclo (delta aminoacyl-L-aminoacyl) (delta aminoacyl = delta Aba, delta Val, delta Leu, delta App, delta Phe and delta Trp; L-aminoacyl=L-Ala, L-Val, L-Leu and L-Lys (epsilon-Ac)) were synthesized by the condensation of cyclo (N-acetyl-Gly-N-acetyl-L-aminoacyl) with corresponding aldehyde or acetone and subsequent treatment with hydrazine. Each cyclo (delta aminoacyl-L-aminoacyl) except ones containing delta Phe and delta Trp was hydrogenated using Pd black in methanol to give cyclo (L-aminoacyl-L-aminoacyl) with high chiral induction. In the case of dehydro-cyclodipeptides containing delta Phe or delta Trp, slightly lower chiral induction was observed. Then, a mechanism of asymmetric hydrogenation was proposed. The influence of variation of solvent, temperature and catalyst on asymmetric hydrogenation was examined. Optically pure L-2-aminobutanoic acid, L-2-amino-5-phenylpentanoic acid and L-phenylalanine were synthesized on a preparative scale by this method and subsequent acid-hydrolysis.

An AM-toxin analog, cyclotetradepsipeptide, containing an O-methyl-L-tyrosine residue in place of an L-2-amino-5-arylpentanoic acid residue in AM-toxins has been prepared by a conventional method. The synthesis was attempted through six different routes and the desired analog was obtained by one such procedure. This compound showed extremely weak activity in causing necrosis on apple leaves.

Several cyclo(-L-aminoacyl-deltaAla-) (aminoacyl = Ala, Val, Leu, Phe, Pro and Lys (epsilon-Ac)) were prepared by tosylation and successive detosylation of cyclo(-L-aminoacyl-L-Ser-), which were synthesized via the Nitecki and Fischer methods. Hydrogenation of the double bond of dehydroalanine residues in cyclodipeptides was carried out using Pd black in methanol at 1-atm pressure and room temperature. The degree of asymmetric hydrogenation was assessed by determining the amounts of L- and D-alanine by a modified Manning and Moore procedure. When L-valine was used as a chiral source, L-alanine residue with chiral induction of 98.4% was derived from cyclo(-L-Val-deltaAla-). L-Amino acids other than L-proline also were effective in inducing remarkable asymmetric hydrogenation. Hydrogenation of alpha,beta-dehydro-alpha-aminobutanoic acid residues in cyclodipeptides produced L-alpha-aminobutanoic acid residues with effective chiral induction to the same extent as observed with dehydroalanine residues. Optically pure l-alanine was prepared from cyclo(-L-Lys(epsilon-Ac)-deltaAla-) via asymmetric hydrogenation. A mechanism of chiral induction is discussed.

To confirm the structure of AM-toxin I (a phytotoxic cyclotetradepsipeptide) the proposed peptide was prepared by a conventional method. The synthetic peptide and natural AM-toxin I were identical as regards t.l.c., u.v., mass spectra and biological activity in causing necrosis on apple leaves. A prepared dimer of AM-toxin I showed extremely weak activity; the relationship between the ring size and biological activity of AM-toxin I is discussed.

Pulmonary functions such as rapid adsorption, respreading, and hysteresis behavior of pulmonary surfactants are very important for respiratory movement. The interfacial behavior of pulmonary preparations containing an amphiphilic peptide (Hel 13-5) has recently investigated. An orientation of hydrophobic chains in a dipalmitoylphosphatidylcholine (DPPC) with or without palmitic acid (PA) is associated with a collapse of alveoli during respiration process. Therefore, the present study focused on the acyl chain orientation in model pulmonary surfactants (DPPC/Hel 13-5 and DPPC/PA/Hel 13-5). A successive change in the orientation during cyclic compression and expansion of films at the air-water interface can be probed directly by an infrared reflection-absorption spectrometry (IRRAS) technique. The hysteresis behavior, one of very important pulmonary functions, was previously observed in surface pressure (pi)-molecular area (A) isotherms for the both model pulmonary surfactant systems (Langmuir 22(2006)1182-1192 and Langmuir 22(2006)5792-5803). In addition, it was reported that Hel 13-5 was squeezed-out of the surface on compression like native pulmonary surfactant proteins. The data obtained for the binary and ternary systems were compared with those of the equivalent pure DPPC and DPPC/PA mixtures, respectively. For an asymmetric methylene stretching vibration (nu(a)-CH(2)) RA intensity, the absolute RA values increased with shifting to small surface area, monotonously. For the corresponding wavenumber, on the other hand, the values gradually decreased into approximately 2920cm(-1). However, they were kept constant in the squeeze-out region in spite of a further decrease of surface area. These results suggested that the orientation of hydrophobic chains in DPPC and DPPC/PA mixtures became in the most packed state soon after emergence of the squeeze-out process of Hel 13-5 and then the packed orientation was retained up to the collapse state. This indicated that the squeezed-out Hel 13-5 stabilized monolayers left at the interface. For the DPPC/PA/Hel 13-5 system, in particular, dissociated PA molecules were excluded together with Hel 13-5 and the surface monolayers were refined to DPPC and undissociated PA components during the compression process. And the similar behavior in the second and third cycles supported the good respreading ability of the monolayers containing Hel 13-5.

Surface pressure (pi)-, surface potential (deltaV)-, and dipole moment (mu(perpendicular))-area (A) isotherms and morphological behavior were examined for monolayers of a newly designed 18-mer amphiphilic alpha-helical peptide (Hel 13-5), DPPC, and DPPC/egg-PC (1:1) and their combinations by the Wilhelmy method, ionizing electrode method, fluorescence microscopy (FM), and atomic force microscopy (AFM). The newly designed Hel 13-5 showed rapid adsorption into the air-liquid interface to form interfacial films such as a SP-B function. Regardless of the composition and constituents in their multicomponent system of DPPC/egg-PC, the collapse pressure (pi(c); approximately 42 mN m(-1)) was constant, implying that Hel 13-5 with the fluid composition of egg-PC is squeezed out of Hel 13-5/DPPC/egg-PC monolayers accompanying a two- to three-dimensional phase transformation. FM showed that adding a small amount of Hel 13-5 to DPPC induced a dispersed pattern of ordered domains with a "moth-eaten" appearance, whereas shrinkage of ordered domains in size occurred for the DPPC/egg-PC mixture with Hel 13-5. Furthermore, AFM indicated that (i) the intermediate phase was formed in pure Hel 13-5 systems between monolayer states and excluded nanoparticles, (ii) protrusions necessarily located on DPPC monolayers, and (iii) beyond the collapse pressure of Hel 13-5, Hel 13-5 was squeezed out of the system into the aqueous subphase. Furthermore, hysteresis curves of these systems nicely resemble those of the DPPC/SP-B and DPPC/SP-C mixtures reported before.

Langmuir monolayer (surface pressure (π)–area (A), surface potential (ΔV)–area (A), and dipole moment (μ⊥)–area (A) isotherms) and fluorescence microscopy techniques were used to investigate a new-designed 18-mer amphiphilic α-helical peptide (Hel 13-5) which consists of 13 hydrophobic and 5 hydrophilic amino acid residues. We present here a study of the surface behavior of Hel 13-5 against dipalmitoylphosphatidylcholine (DPPC) and/or Egg-phosphatidylcholine (Egg-PC), which are major components in artificial pulmonary surfactant. A temperature dependence of pure DPPC and Hel 13-5 was examined using Langmuir isotherm techniques over the temperature range of 298.2–310.2 K. Basic interfacial behavior of Hel 13-5 was investigated by adding Hel 13-5 to pure DPPC and the DPPC/Egg-PC (1:1, mol:mol) mixture on a substrate solution of 0.02 M Tris buffer (pH 7.4) with 0.13 M NaCl at 298.2 K. The cyclic compression–expansion isotherms of these systems were obtained to confirm the spreading and respreading abilities of Hel 13-5. In addition, fluorescence microscopy measurements were carried out to understand the interactions of Hel 13-5 with pure DPPC or the DPPC/Egg-PC mixture. From the fluorescent images, the distinct differences between these systems were observed. Adding a small amount of Hel 13-5 to DPPC induced the “moth-eaten” disaggregation of liquid-condensed (LC) domains made of DPPC. On the other hand, the phenomenon that the LC domains were shrunk in size by adding a small amount of Hel 13-5 occurred for the there-component system (DPPC/Egg-PC/Hel 13-5).

In order to investigate the influence of cholesterol (Ch) and monosialoganglioside (GM1) on the release and subsequent deposition/aggregation of amyloid beta peptide (Abeta)-(1-40) and Abeta-(1-42), we have examined Abeta peptide model membrane interactions by circular dichroism, turbidity measurements, and transmission electron microscopy (TEM). Model liposomes containing Abeta peptide and a lipid mixture composition similar to that found in the cerebral cortex membranes (CCM-lipid) have been prepared. In all, four Abeta-containing liposomes were investigated: CCM-lipid; liposomes with no GM1 (GM1-free lipid); those with no cholesterol (Ch-free lipid); liposomes with neither cholesterol nor GM1 (Ch-GM1-free lipid). In CCM liposomes, Abeta was rapidly released from membranes to form a well defined fibril structure. However, for the GM1-free lipid, Abeta was first released to yield a fibril structure about the membrane surface, then the membrane became disrupted resulting in the formation of small vesicles. In Ch-free lipid, a fibril structure with a phospholipid membrane-like shadow formed, but this differed from the well defined fibril structure seen for CCM-lipid. In Ch-GM1-free lipid, no fibril structure formed, possibly because of membrane solubilization by Abeta. The absence of fibril structure was noted at physiological extracellular pH (7.4) and also at liposomal/endosomal pH (5.5). Our results suggest a possible role for both Ch and GM1 in the membrane release of Abeta from brain lipid bilayers.

Protein engineering is an emerging area that has expanded our understanding of protein folding and laid the groundwork for the creation of unprecedented structures with unique functions. We previously designed the first native-like pore-forming protein, small globular protein (SGP). We show here that this artificially engineered protein has membrane-disrupting properties and anti-tumor activity in several cancer animal models. We propose and validate a mechanism for the selectivity of SGP toward cell membranes in tumors. SGP is the prototype for a new class of artificial proteins designed for therapeutic applications.

Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex protein-lipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic α-helical peptide, Hel 13-5, transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question.

To study thermodynamically the so-called lowering effect of the plasma cholesterol level caused by dietary intake of phytosterols (plant sterols) or phytostanols, solubilization was investigated for the respective single systems of cholesterol (Ch), cholestanol (Chsta), and stigmasterol (Stig) and for their 1:1 mixed systems with two kinds of free bile salts (BS), sodium cholate (NaC) and sodium deoxycholate (NaDC), in pH 10 Kolthoff buffer solution at 37 °C. For the sterol/stanol solubilizates examined, compared with NaC, NaDC is stronger in solubilizing power, Sp, which is defined as the derivative of the solubilized amount, W, in mM by the total BS concentration, Ct (dW/dCt), as expected in terms of hydrophobicity and aggregation number. The following findings were made: (i) the amount solubilized by both BS micelles follows the order Ch > Chsta > Stig among the three single solubilizates; (ii) Chsta is much stronger in lowering ability than Stig in regard to Ch solubilization; (iii) from the 1:1 mixt...

A 26-mer peptide with the sequence of the pore forming region (residues 427–452) of the Shaker K + channel (H5 region) was chemically synthesized. Analyses by CD and two-dimensional 1 H NMR spectroscopy were used to investigate the structure of the peptide bound to SDS micelles in solution, which are commonly used in biophysical studies. The tertiary structure of the peptide as a monomer was composed of an α-helix (431–438), a turn (439–442), and random coils (427–430, 443–452), and was very similar to that of the pore forming region of the native K + channel from Streptomyces lividans determined by X-ray analysis [1] . This result suggests that even an isolated peptide forms a native-like conformation for residues from 431 to 442, depending on its intrinsic amino acid sequence and the surrounding environment.

In order to investigate the structure-activity relationships between the hydrophobicity, differences in the number of positive charged amino acids and D-amino acids as well as their action mechanism with different bio-membranes, we designed and synthesized two sets of 12-mer model peptides originating from an amphiphilic antimicrobial model peptide and a D-amino acid-containing non-amphiphilic peptide. The introduction of D-amino acid in peptides led not only to decreases in the hydrophobicity, helical content, and hemolytic activity, but to an increase in the antimicrobial activity against Gram-positive and -negative bacteria. The relative peptide hydrophobicity was estimated in terms of the peptide elution time, expressed by linear trifluoroacetic acid (TFA)-water to TFA-acetonitrile gradients on C18 reverse-phase high-performance liquid chromatography (RP-HPLC). The retention behavior of model peptides on RP-HPLC is correlated with the activity and selectivity for mammalian cells, Gram-positive and -ne...

Temperature studies were performed on the micelle formation of a series of anionic surfactants, i.e. Na salts of α-sulfonatomyristic acid methyl, ethyl, and propyl esters (abbreviated as α-SMy·Me, ...

A de novo designed pore-forming small globular protein (SGP) with antitumor activity consists of four helices: 3 basic amphipathic helices composed of Leu and Lys surrounding a central hydrophobic helix composed of oligoalanine. These helices are connected by a β-turn-forming sequence and two β-turn-unfavorable ones (S. Lee, T. Kiyota, T. Kunitake, E. Matsumoto, S. Yamashita, K. Anzai, and G. Sugihara Biochemistry 1997, Vol. 36, pp. 3782–3791). In the present work, we designed and synthesized three new SGP analogs in order to study the stoichiometric packing geometry and stability of SGP. The replacement of alanines in the central helix of SGP with leucines (SGP-L), which make the helix much larger in size and more hydrophobic, resulted in an equilibrium of monomeric–trimeric structure. The replacement of some Lys residues by Glu residues in the hydrophilic regions of the amphipathic helices (SGP-E) led to a decrease in helical content and the formation of an equilibrium of monomeric–trimeric structure. The alteration of β-turn regions with Gly residues, which makes these regions flexible (SGP-G), established an equilibrium of monomeric–dimeric states in buffer. The hydrophobic α-helix of SGP-L penetrated into the lipid bilayers in a manner that stabilized model membranes and biomembranes, whereas the central helices of SGP-G and -E destabilized them by forming channels. SGP and its analogs may be a useful model to study the role of the hydrophobic and hydrophilic regions in the formation of monomer–oligomer of proteins and to better understand the insertion of membrane targeting proteins into biomembranes. © 2001 John Wiley & Sons, Inc. Biopolymers 56: 96–108, 2001

To study the formation of micelles in bulk water and adsorbed film at the air/water interface, we have thermodynamically studied a novel type of anionic surfactant, the so-called “hybrid type”. We used sodium 1-oxo-1[4-(tridecafluorohexyl)phenyl]-2-hexane sulfonate (here, abbreviated as FC6-HC4), which involves a perfluorocarbon chain attached to the phenyl group as the primary hydrophobic group and a short hydrocarbon side chain attached to a sulfonate group.The surface tension (γ) of solutions at different temperatures was measured by means of a drop volume technique. The critical micellization concentrations (cmc's) were determined from the plot of γ vs logarithmic molality as a function of temperature ranging from 5 to 50 °C and the cmc−temperature curve was found to have a minimum of ca. 15 °C. The degree of counterion binding (β) was estimated at each temperature from the Corrin−Harkins plot. The β value itself shows a little temperature dependency ranging around 0.57, which is smaller than those of...

The adsorption isotherms of various free and conjugated bile salts (BS) onto the surface of graphite (Gr) powder in aqueous borate buffer solution at pH 10 were obtained as a function of temperature ranging from 20 or 25 to 42 °C. Analysis by Langmuir plot was made within the concentration range where the Langmuir adsorption took place, and from it the maximum amount of monolayer adsorption (Nm) and the Langmuir constant (KL), i.e., the ratio of rate constants of adsorption and desorption (KL = ka/kd), were determined for each BS at different temperatures. The present study shows the KL to be the reciprocal of equilibrium concentration (C*) at half surface coverage, as KL = 1/C*, and in addition, KL is equal to the adsorption equilibrium constant, Kad, which was defined by a thermodynamic consideration. For determination of KL or Kad, a more reliable method was proposed by to more easily examine the accuracy of the measured points. From the van't Hoff plot of Kad, the enthalpy changes (adsorption heats, Δ...

In a work published in this journal by J.P.S. Cabral and A.R.W. Smith [J. Colloid Interface Sci. 149 (1992) 27], it is reported that the 1-dodecylguanidinium acetate (dodine) exhibits first CMC at 20-30 microM and second CMC at 110-120 microM in aqueous solution at 22.5 degrees C. Such low CMCs are unusual for ionic surfactants with dodecyl chain, and is quite interesting if this is the case. Thus, we investigated the micelle formation of dodine by electrical conductivity measurements. The specific conductivity, kappa, vs concentration plot showed no evidence for micelle formation up to a few hundreds microM at 25 degrees C. The Krafft temperature of dodine was found to be approx. 52 degrees C. When conductivity measurements were made at 54 degrees C, a clear break point was observed in the kappa vs concentration plot at 9.5 mM, which must correspond to the CMC of dodine. This CMC value is quite normal for cationic surfactant with dodecyl chain.

The conformations of synthetic peptides corresponding to the first membrane-contact portion from Tyr390 to Lys430 of band 3 (band 3-1a) and the counterpart portion of South-East Asian ovalocytosis (SAO) band 3 (band 3-1b) in lipid bilayers were examined by means of circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy as well as a proteolytic digestion method. The CD and FTIR studies showed that band 3-1a and band 3-1b in a membrane lipid bilayer cannot assume an alpha-helix rich structure but instead assume a beta-structure rich conformation. The proteolytic digestion experiments demonstrated that the cleavage sites of Tyr392 and Phe423 were common to both the model and erythrocyte membranes. Taken together with our previous work, which indicated that the first membrane-contact portion was the portion embedded in the erythrocyte membrane without tight lipid-peptide interactions [Hamasaki et al. (1997) J. Biochem. 122, 577-585], we imply herein that the first membrane-contact portion of band 3 by itself can not assume the ordinary alpha-helix conformation in the membrane lipid bilayers. A proteinase-resistant portion, from Ser402 to Phe423, was observed when liposomes containing band 3-1a were digested with proteinase K, while no proteinase-resistant core portion was found in the case of band 3-1b (DeltaAla400-Ala408). This suggests the crucial role of the deleted portion, from Ala400 to Ala408, in the interaction of the first membrane-contact portion of band 3 with a membrane lipid bilayer.

We previously designed and synthesized five N- and C-termini-free amphiphilic α-helical model peptides (Hel series) with a systematically varied hydrophobic–hydrophilic balance (HHB) that showed hemolytic activity, but no antimicrobial activity. However, an N-acetylated and C-amidated model peptide, peptide 3 [S. E. Blondelle and R. A. Houghten, Biochemistry, 31, 12688 (1992)], similar to a Hel series peptide, Hel 9-9, whose hydrophobic and hydrophilic amino acid residues and areas are equal in the α-helical structure, have exhibited both hemolytic and antimicrobial activities. Thus, to investigate the N- and C-terminal effect of the Hel series peptides on their antimicrobial activity, we designed and synthesized three peptides (Cap-Hel series), both termini-blocked by N-acetyl and C-amide groups. Their interaction mode with membranes was examined through reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy as well as measurements of the hemolytic activity, antimicrobia...

It has been showned that cationic α-helical peptides can be useful as nucleic acid-carrier molecules for gene transfer into cells. In order to investigate the significancemake sure of importance of the hydrophobic region in amphiphilic peptides in relation to their transfection ability, we have employed five kinds of peptides with a systematically varied hydrophobic-hydrophilic balance in the amphiphilic structures, and have evaluated the relationship between the structure and the gene transfer ability of the peptides into COS-7 cells. The peptides with a large hydrophobic region took α-helical structures, formed large aggregates and showed high transfection efficiency. Their high efficiency can be explained on the basis of their ability to form stable aggregates which can be internalized by endocytosis and remain resistant to digestion in lysosomal vesicles. Furthermore, it was suggested that the hydrophobic region of peptides plays an important role in the disruption of the endosomal membrane, which ca prevent the degradation of DNA in lysosomal vesicles. When peptides do not have so strong membrane-disruptive activity, but form aggregates which can be incorporated by endocytosis, the transfection efficiency can be recovered by the addition of an endosome-disruptive peptide.

In order to make clear the adsorption mechanism as well as the effective hydrophobicity of four kinds of sodium salts of bile acids (cholate (NaC), deoxycholate (NaDC), chenodeoxycholate (NaCDC) and ursodeoxycholate (NaUDC)), the effect of temperature on the adsorption on graphite (12.9 m 2 g −1 ) was examined for these bile salts in tetraborate-carbonate buffer solution (at pH 10.0). The isotherms observed exhibit a two-step adsorption below 30°C, but above 37°C monolayer adsorption takes place, suggesting that there exists a certain transition temperature. In some cases a further increase in adsorption, of a BET-type, was observed at higher concentrations. From the Langmuir plot, the maximum adsorption amounts of monolayer, N m , the constants of adsorption equilibrium, b , and the mean surface areas, A m , occupied by a bile salt molecules were determined as a function of temperature (at 25, 30, 37 and 42°C). The van't Hoff plot was applied to estimate the heat of adsorption, − ΔH , of each bile salt on graphite. The decreasing orders of − ΔH and N m , were found to be NaCDC>NaC>NaDC>NaUDC, and NaDC>NaCDC>NaC>NaUDC, respectively. The disagreement between the orders is discussed.

The mode of interaction of two pyrenylalanine residues which are introduced at positions 1 and 3′ into gramicidine S, [Pya 1,3′ ]GS, was investigated through absorption, CD, and steady-state and time-resolved fluorescence spectroscopy in organic and buffer solutions. CD spectra showed that the pyrene moieties could adopt an orientation to form a sandwich-type close pair on the two strands in a typical antiparallel β-structure in trifluoroethanol. Other spectroscopic studies demonstrated that the pyrenes of [Pya 1,3′ ]GS in trifluoroethanol formed specific intramolecular association on the pyrene pair, a ‘sandwich-type dimer’, in the ground state.

Tachyplesin I, an antibacterial and antiviral heptadecapeptide from the hemocyte debris of Tachypleus tridentatus, contains four aromatic amino acids (Trp2, Phe4, Tyr8, and Tyr13) which have been shown to be crucial for activity. In order to investigate conformations and orientations of the aromatic amino acid residues of tachyplesin I in lipid bilayers, its analogs, [Phe8]- and/or [Phe13]-tachyplesin(s) I in which Tyr8 and Tyr13 are replaced by Phe, were synthesized. Circular dichroism spectral studies showed that three peptides are considerably different in conformation in aqueous solution at pH 8.0 whereas they take similar conformations in the presence of neutral egg yolk phosphatidylcholine (EYPC) liposomes. Energy transfer kinetics showed that the distances of Trp2-Tyr8 and Trp2-Tyr13 are 16 A (max of 18.3 A, min of 15.1 A) and 18 A (max of 20.2 A, min of 16.6 A) in buffer but are 12 A (max of 15.2 A, min of 8.6 A) and 18 A (max of 22.9 A, min of 12.9 A), respectively, in the presence of acidic EYPC/EYPG (3:1) liposomes. Decay kinetics for Trp2 fluorescence indicated that Trp2 takes at least three conformations in buffer and in acidic liposomes where fractions of three Trp2 conformers vary by changing the medium from buffer to acidic liposomes. Although tachyplesin I is not in amphiphilic structure in buffer in spite of its rigid antiparallel beta-conformation, its interaction with lipid bilayers appears to induce amphiphilic structure via minor alteration of peptide backbone and side chain orientations, resulting in shortening the distance of Trp2-Tyr8.

Micelle formation and adsorption at air/water interface were investigated as function of temperature for aqueous solutions of the nonionic surfactant, n-nonyl-β-D-thiomaltoside (NTM), used as a membrane-protein solubilizer by surface tension measurements (drop volume method). For comparison with NTM, such surface tension measurements were performed even for aqueous solutions of decanoyl-N-methylglucamide (MEGA-10). Examination was also made of the effect of adding alcohols (methanol, ethanol and 1-butanol) on critical micellization concentration (cmc) and on surface activity. As for the behavior of surface activity as well as cmc, NTM was found more similar to ionic surfactants than typical nonionic surfactants such as poly oxy ethylene alkyl ethers ; NTM exhibited no cloud point at least up to 100°C, but showed a minimum cmc at 28.3°C in the curve of temperature vs. cmc. Light scattering measurement at 25°C (the Debye plot) gave a comparatively small aggregation number of 16.4, indicating that the present thermodynamic analysis based on the phase separation model (PSM) may involve a rough approximation. However, a thermodynamical feature of NTM may be understood from PSM and facilitates comparison with MEGA-10 and alkyl glucosides.In addition, the hydrophilic-lipophilic balance (H.L.B.) value not indicated by Davies was determined for sulfur in amphiphilic organic compounds (-S-) as 1.5.

Surface pressure (π) and surface potential (Δ V )–area ( A ) isotherms were obtained for mixed monolayers of different perfluorocarboxylic acids (FCs) with a hydrocarbon fatty acid, tetradecanoic acid (HC14), on a substrate solution of pH 1.0 as a function of compositions in the mixture by employing the Langmuir method and the ionizing electrode method. Data for the mixed monolayers were analyzed in terms of the additivity rule. Judging from Matuo's classification of two-dimensional phase diagrams, our phase diagrams might be classified into three types. The first is a positive azeotrope type; the combinations of tetradecanoic acid (HC14) with perfluorodecanoic acid (FC10) and with perfluorododecanoic acid (FC12) seem miscible with each other. The second is a completely immiscible type; the combination of perfluorotetradecanoic acid (FC14) and tetradecanoic acid. The third might be a eutectic type, to which the combinations of HC14 with FC16 and with FC18 are assigned; they are miscible in the expanded state but immiscible in the condensed state. Furthermore, the data on the mean molecular area, the surface dipole moment, and the phase diagrams constructed enabled us to estimate the molecular orientation of the respective acids in the mixed monolayer state.

The cationic derivatives, the hydrochloride salts of ethylenediamine monodeoxycholate (EdaDC) and cholate (EdaC) derived from the respective bile salts, sodium deoxycholate (NaDC) and cholate (NaC), were synthesized and their physicochemical and biological properties together with the possibility of application as crystallizing agents for membrane proteins were studied. The critical micelle concentrations (CMCs) in water were determined to be 4.0 mM for EdaDC and 9.0 mM for EdaC, and the surface tensions were lowered down to 43 and 41 mN m−1, respectively, in the range above CMCs. When compared with the corresponding natural bile salts, the two cationic bile acid derivatives have the highest surfactant ability, followed by the corresponding taurine-conjugated salts of TDC (taurodeoxycholic acid) and TC (taurocholic acid) and the free bile acid salts of DC and C have the lowest activity. Studies of dye release from pre-encapsulated vesicles, hemolysis and antimicrobial activity indicated that the concentrations leading to a large increase in the membrane permeability and biomembrane perturbation ability are signiicantly lower than the CMCs of these surfactants. Buffer solution containing EdaC or EdaDC could solubilize a membrane protein, light-harvesting chlorophyll ab protein complex (LHCII), at concentrations higher than CMCs, but the power was estimated to be one third of that of surfactants such as Triton X-100, n-octyl-β-d-glucopyranoside and n-nonyl-β-d-glucopyranoside. Fluroescence depolarization studies showed that some specific molecular interactions between LHCIIs are induced by the cationic bile salts, suggesting that the latter would be valid for the crystallization of LHCII. In fact, LHCII crystals were observed in crystallization media containing 1% EdaC or 0.5% EdaDC.

Monolayers formed by glycine and taurine conjugates of chenodeoxycholic acid (GCDC and TCDC respectively) and ursodeoxycholic acid (GUDC and TUDC respectively) and cholesterol (Ch), and, in addition, those formed by different binary mixed systems of these bile acids (BAs) with Ch (GCDC/Ch, GUDC/Ch, TCDC/Ch and TUDC/Ch) were investigated in terms of surface pressure (π), surface potential (ΔV) and surface dipole moment (μ⊥) as a function of average surface (occupation) area (A). On the substrate of 5 M aqueous NaCl solution at pH 1.2 and 25°C, the π-A curves demonstrated that Ch forms a typical condensed monolayer film whereas each conjugated BA forms a nontypical expanded film which is clearly different from a typical one formed by free BAs such as deoxycholic acid (DC) and chenodeoxycholic acid (CDC). Every mixed system showed that its π-A, ΔV-A and μ⊥-A curves are inserted inbetween those of the respective pure systems in a regular sequence of mole fraction of one component in the binary mixture. All the mixed systems of BA with Ch were found to be completely immiscible with each other and to form a “patched monolayer” on the substrate, and this led us to conclude that compliance with the additivity rule as for A, ΔV or μ⊥ is a necessary, but not wholly sufficient, condition for ideal mixing in the two-dimensional phase.

Monolayers formed in 5 M aqueous NaCl solution at pH 1.2 and 25°C were studied for glycine and taurine conjugates of chenodeoxycholic acid (GCDC and TCDC) and ursodeoxycholic acid (GUDC and TUDC) and two mixed binary systems of these bile acids (BA), i.e. GCDC/GUDC and TCDC/TUDC, being examined in terms of surface pressure (π), surface potential ( ΔV ) and surface dipole moment ( μ ⊥ ) as a function of average surface (occupation) area ( A ). The π- A curves demonstrated that all the conjugated BAs studied form a non-typical expanded film which is clearly different from a typical one formed by free BAs such as deoxycholic acid and chenodeoxycholic acid. It was found for these conjugated BAs that with increasing μ ⊥ the compressibility of the monolayer is reduced, and beyond the maximum of μ ⊥ at which the π- A curve has an inflection point, the compressibility is enhanced with compression (in other words, the elasticity of the film increases and then, via a maximum, decreases with decrease in surface area), leading finally to collapse of the liquid film. The minimum compressibility ( δ min ) was evaluated for each mixed system as a function of composition. The constructed two-dimensional phase diagrams of GCDC/GUDC mixture and TCDC/TUDC mixture revealed that the former and latter mixed systems both form homogeneously mixed monolayers but deviate negatively and positively respectively from ideal mixing.

The adsorption behavior on graphite was examined for four kinds of sodium salts of bile acids; deoxycholate (NaDC), chenodeoxycholate (NaCDC), ursodeoxycholate (NaUDC), and cholate (NaC) in tetraboratecarbonate buffer solution (at pH 10.0) mainly at 30°C. All the salts seemed to exhibit the Langmuir type isotherm (plots of adsorption amount vs. equilibrium concentration), but when examined in more detail a small step was observed in each isotherm, suggesting that a certain phase transition or adsorption onto the first monolayer might take place. All the isotherms obtained enabled us to construct both the Langmuir plot and the BET plot. From both plots the maximum amounts of monolayer adsorption, N m , the constants of adsorption equilibrium, K , and the mean surface areas, A m , occupied by a bile salt molecule were determined; N m decreases in the order NaDC, NaCDC, NaC and NaUDC. These data were compared with the results of previous work on the adsorption behavior of these four bile salts at the cholesterol (Ch) crystal/water interface (the order of N m on Ch crystals is NaDC > NaCDC > NaUDC > NaC). The adsorption behavior of each bile salt was discussed in terms of the hydrophobic indexes (HI), the limiting surface area, A 0 , determined from the previous monolayer study, the solubilizing power (Sp) for cholesterol, and so on.

Mixed monolayer formation of deoxycholic acid (DC), a typical free bile acid (BA), with cholesterol (Ch) was studied in terms of the relations of average molecular (occupation) area (A) with surface pressure (π), surface potentia (ΔV) and surface dipole moment (μ⊥) as a function of mole fraction of Ch in the binary mixture. Although the additivity rule was perfectly satisfied for π, ΔV and μ⊥, the present mixed system showed that DC cannot mix with Ch in the monolayer; instead, they form a “patched monolayer”, indicating the lack of lateral intermolecular interaction between Ch and BAs.

Mastoparan B (MP-B), an amphiphiphilic α-helical peptide newly isolated from the hornet Vespa basalis, was studied in comparison with mastoparan (MP), in terms of interaction with the phospholipid bilayer and of hemolytic and antimicrobial activity. The amphiphiphilic structure of MP-B has more hydrophilic amino acid residues in the hydrophilic surface than that of MP. Although each peptide had a considerably different effect on the interaction with lipid bilayers (e. g., their conformation in the presence of acidic and of neutral lipids and dye-release ability from the encapsulated liposomes), on the whole the interaction mode was similar. MP-B caused a change in the shape of erythrocytes from normal discoid to a crenated form (named echinocytes). MP exhibited strong activity against gram-positive bacteria but not against gram-negative ones. Contrary to this, MP-B showed both strong activity against gram-positive bacteria and potent activity against gram-negative bacteria. Whereas both peptides have almost the same residues on the hydrophobic side, the difference in the hydrophilic surface area on the molecules seems to lead to the subtle change in its interaction with membranes, resulting in the alteration of biological activity. © 1995 John Wiley & Sons, Inc.

In order to investigate the role of each amino acid residue in determining the secondary structure of the transmembrane segment of membrane proteins in a lipid bilayer, we made a conformational analysis by CD for lipid-soluble homooligopeptides, benzyloxycarbonyl-(Z-)Aaan-OEt (n = 5 - 7), composed of Ala, Leu, Val, and Phe, in three media of trifluoroethanol, sodium dodecyl sulfate micelle, and phospholipid liposomes. The lipid-peptide interaction was also studied through the observation of bilayer phase transition by differential scanning calorimetry (DSC). The CD studies showed that peptides except for Phe oligomers are present as a mainly random structure in trifluoroethanol, as a mixture of alpha-helix, beta-sheet, beta-turn, and/or random in micelles above the critical micellization concentration and preferably as an extended structure of alpha-helical or beta-structure in dipalmitoyl-D,L-alpha-phosphatidylcholine (DPPC) liposomes of gel state. That the beta-structural content of Val oligomers in lipid bilayers is much higher than that in micelles and the oligopeptides of Leu (n = 7) and Ala (n = 6) can take an alpha-helical structure with one to two turns in lipid bilayers despite their short chain lengths indicates that lipid bilayers can stabilize the extended structures of both alpha-helical and beta-structures of the peptides. The DSC study for bilayer phase transition of DPPC/peptide mixtures showed that the Leu oligomer virtually affects neither the temperature nor the enthalpy of the transition, while Val and Ala oligomers slightly reduce the transition enthalpy without altering the transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to identify the respective roles of bile salts (which are different depending on the species) in the solubilization of cholesterol (Ch) or in the dissolution of Ch-type gallstones, the adsorption behavior of bile salts on a Ch crystal surface in aqueous media was examined using four kinds of sodium salts: the deoxycholate (NaDC), the chenodeoxy-cholate (NaCDC), the ursodeoxycholate (NaUDC), and the cholate (NaC), mainly at 37°C. All the salts studied exhibit a two-step adsorption isotherm (assigned to type IV according to Brunauer's classification), but regarding the adsorption amount, these four bile salts are divided into two groups; the group comprising NaDC and NaCDC has a higher ability to adsorb at the Ch/water interface than the other group which comprises NaUDC and NaC. From the BET plot and/or the Langmuir plot, the maximum amounts of monolayer adsorption, the constants of adsorption equilibrium and the mean surface areas occupied by a bile salt molecule were determined. The amount of monolayer adsorption decreases in the order NaDC, NaCDC, NaUDC and NaC. These data were discussed in relation to the CMC, the hydrophobic indexes of the α and β sides of the bile acid molecule, and the solubilizing power (the molar ratio of solubilized cholesterol to bile salt). The species dependence of Ch solubilization was interpreted in terms of hydrophobic room space in a micelle constructed with aggregated bile salts.

The critical micelle concentration (cmc) and the degree of counterion binding (β) to micelles were determined as a function of composition in the mixture of dodecylammonium perfluoroacetate (DAPA) with dodecylammonium perfluoropropionate (DAPP) in water at 40°C. The curve of cmc vs XDAPP (the mole fraction of DAPP in the surfactant mixture) was finely simulated by Rubingh's equations, which enabled us to estimate the interaction parameter (WR) based on the approximation of the regular solution; the curve fitting exhibited a slightly negative value (WR = -0.131), indicating that the mixing enhances micelle formation due to a greater interaction than those of the respective pure systems. On the other hand, to the same mixed system were simultaneously applied our theoretical equations which were derived by taking into account the counterion effect. In contrast to the result estimated from Rubingh's equations, the latter analysis indicated that the mixing of DAPA with DAPP seemed to be ideal, i.e., the interaction parameter (W0) was estimated to be nearly equal to zero. The difference between WR and W0 is directly ascribable to the contribution of counterions to the free energy of mixed micelle formation. The measured β values were in an exact agreement with those calculated from the micellar composition estimated on the basis of our theory.

A temperature study was performed on micelle formation of a series of homologous cationic surfactants having organic counterions (alkanesulfonates) with carbon numbers ranging from 1 to 4: dodecylammonium salts of methanesulfonate (DAMS), ethanesulfonate (DAES), propanesulfonate (DAPS), and butanesulfonate (DABS) in water. The critical micelle concentrations (CMCs) and the degree of counterion binding (β) were determined at different temperatures ranging from 5 to 50°C by means of conventional electric conductance measurements. From the temperature dependence of β as well as CMC, Gibbs energy ΔG0m, enthalpy ΔH0m, and entropy ΔS0m, on micelle formation, were estimated for the respective surfactants. As for the temperature dependence of CMC for these surfactants, the temperature-CMC curves have a minimum around 30°C and show that the CMC at each temperature is lowered by about 3 mmol dm-3 per methylene group in the alkyl chain of the counterions. The relationship between β and temperature suggested that the counterion of MS- behaves most similarly to common univalent ions such as halide ions. In contrast, PS- and BS-, having a stronger ability to lower CMC and to promote association of surfactant ions with counterions as well as of surfactant ions themselves, behave more like those of surfactant ions, and ES- shows the most complicated character between those of common univalent ions and organic ions. However, the temperature dependence of enthalpy change, ΔH0m demonstrates that these four surfactants are divided into two groups: (1) DAMS and DAES and (2) DAPS and DABS. In addition, the entropy change ΔS0m as a function of alkyl chain length gives evidence that the contribution of the entropy term to the Gibbs energy on micelle formation clearly separates between DAES (m = 2) and DAPS (m = 3). A similar discontinuity is found even in the plot of ΔG0m versus carbon atom number of alkyl chain, m, and in the plot of ΔG0m versus estimated hydrodynamic radius of counterions. All the results obtained have indicated that lengthening the alkyl chains initially hinders micelle formation, but the longer chains are markedly effective in lowering the CMC and probably in increasing the aggregation number, owing to enhanced hydrophobic interaction between counterion and the micellar surface and/or core.

In order to elucidate the roles of the N-terminal segments of groups I and II phospholipases A2 (PLA2s) which have been known to have alpha-helical structure and have been assumed to be involved in the water/lipid interface recognition site, the peptides corresponding to the N-terminal moieties of group I PLA2 (Naja naja atra) and group II PLA2s (Trimeresurus flavoviridis and Crotalus atrox) were synthesized and their interactions with model membranes were studied. Circular dichroism spectra showed that N-terminal peptides of both groups I and II PLA2s took alpha-helical structure in trifluoroethanol but no significant secondary structure in buffer (pH 8.0). In the presence of acidic liposomes, N-terminal fragments of group II PLA2s formed alpha-helical structure, while that of group I PLA2 remained unaffected. The hydrophobic moments showed that amphipathicities of N-terminal fragments of group II PLA2s are evidently larger than those of N-terminal fragments of group I PLA2s. The leakage of carboxyfluorescein from acidic liposomes was induced only with group II PLA2 peptides. Large blue shift and increase in intensity of tryptophan fluorescence were also observed for group II PLA2 peptides when interacting with acidic liposomes. Such difference in the modes of interactions with lipid bilayers between N-terminal peptides of groups I and II PLA2s appears to be due in large part to the difference in intrinsic alpha-helix forming properties of their amino acid sequences. It is inferred that N-terminal amphipathic alpha-helical structures of group I PLA2s are possibly formed by assistance of a neighboring chain bridged by Cys-11 and Cys-77.

It has been reported that a peptide corresponding to the S4 segment in sodium channel protein is able to form voltage-dependent cation-selective ion channels (Tosteson, M. T., Auld, D. S., and Tosteson, D. C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 707-710). However, biological and other physical properties remain unexamined. In the present study, three peptides, H-(Ala-Arg-Leu)8-OH (ARL8), H-(Val-Arg-Leu)8-OH (VRL8), and H-(Leu-Arg-Leu)8-OH (LRL8) which were designed on the basis of the S4 segment and expected to form 3(10)-helix, were synthesized and examined with regard to conformational change by the interaction with membranes, membrane perturbation ability, ion channel formation, and antimicrobial activity. According to CD spectra, these peptides were found to form a 3(10)-helical structure in the presence of dipalmitoyl-DL-alpha-phosphatidylcholine/dipalmitoyl-DL-alpha- phosphatidylglycerol (3:1) liposomes. The experiment of the peptide-induced leakage of carboxyfluorescein from liposomes showed that all the peptides had a strong ability to perturb membranes. The peptides were able to form cation-selective ion channels in planar asolectin lipid bilayers. The conductances of the ion channels were small (approximately 2 picosiemens for VRL8 and LRL8 and approximately 23 picosiemens for ARL8), suggesting that the peptides produce narrow pores or wider pores with certain permeable barriers that are a portion of the whole channels. The differences in their conductances depend possibly on the sizes of the side chains of Ala, Val, and Leu residues. However, non of the peptides showed antimicrobial activity (minimum inhibitory concentrations, > 50 micrograms/ml). Here, we present the first evidence that the peptides can form 3(10)-helical structures with long chain lengths in a lipid bilayer environment.