Five amphiphilic alpha-helical peptides of 18 residues containing a hydrophobic Trp residue as a fluorescence probe were designed. The peptides were made up of hydrophobic Leu and hydrophilic Lys residues of a ratio of 13:5, 11:7, 9:9, 7:11, and 5:13 (abbreviated as Hels 13-5, 11-7, 9-9, 7-11, and 5-13, respectively). These peptides generate ideal amphiphilic alpha-helical structures, which have systematically varied hydrophobic-hydrophilic balance (relative amphiphilic potential) as a result of different hydrophobicities and almost the same hydrophobic moments. Their hydrophobic-hydrophilic balance was estimated both theoretically from the calculated hydrophobicity values (or the magnitude of hydrophobic faces) and experimentally from the retention times in reverse phase high-performance liquid chromatography (RP-HPLC). Circular dichroism, liposome-lytic, and Trp-fluorescent studies in buffer and in the presence of acidic and neutral liposomes clearly showed that the increasing hydrophobic face area not only increases the affinity for lipid but also increases the trend of self-association. The structure-activity relationship estimated by means of leakage ability and hemolytic activity demonstrated that the model- and bio-membrane perturbation ability is completely parallel to the magnitude of the hydrophobic face area. The lipid-binding study in guanidine hydrochloride solution showed that the peptides with a hydrophobic face larger than the hydrophilic face (Hels 13-5 and 11-7) immerse their hydrophobic regions in lipid bilayers and that the inverse ones (Hels 7-11 and 5-13) interact only between the anionic lipid head groups and cationic peptide residues on liposome surfaces. The peptide Hel 9-9, which has exactly the same hydrophobic and hydrophilic regions, was found to be at a critical boundary among these peptides in terms of (1) behavior of peptide self-aggregation in buffer solution and membrane perturbation ability, (2) transfer from bulk solution to neutral lipid bilayers, and (3) necessity of charge interaction in lipid-peptide binding.

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.

Mastoparan B (MP-B), an amphiphilic alpha-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 amphiphilic 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.

In order to investigate the influence of cholesterol (Ch) and monosialoganglioside (GM1) on the release and subsequent deposition/aggregation of amyloid ␤ peptide (A␤)-(1-40) and A␤-(1-42), we have examined A␤ peptide model membrane interactions by circular dichroism, turbidity measurements, and transmission electron microscopy (TEM). Model liposomes containing A␤ peptide and a lipid mixture composition similar to that found in the cerebral cortex membranes (CCM-lipid) have been prepared. In all, four A␤-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, A␤ was rapidly released from membranes to form a well defined fibril structure. However, for the GM1-free lipid, A␤ 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 A␤. 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 A␤ from brain lipid bilayers. The pathology of Alzheimer's disease (AD)1 includes extracellular amyloid plaques, intraneuronal neurofibrillary tangles, synaptic loss, and neuronal cell death. The major components of amyloid plaques are the amphiphilic 40 and 42 residue peptides, A␤-(1-40) and A␤-(1-42) (1, 2). Amyloid ␤-peptide (A␤) consists of a hydrophilic N-terminal region (residues 1-28) and a hydrophobic C-terminal region (residues 29 -40 or 29 -42). The hydrophobic part of A␤ is originally part of a transmembrane ␣-helix of APP anchored in the membrane of several subcellular compartments, including the ER (3). Proteolysis by the enzyme(s) ␥-secretase leads to the formation of A␤ within the membrane. Thus, the membrane release of A␤ following this enzyme cleavage should play a pivotal role in subsequent amyloid plaque formation.Recent studies have shown that the interaction of A␤ and lipids plays an important role in the pathogenesis of AD. For instance, the fibrillogenic properties of A␤ are in part a consequence of the composition of the membrane in which it resides, its peptide sequence, and its mode of assembly within the membrane (4). In terms of membrane composition, Ch and GM1 in neuronal cell membranes are widely accepted to be modulators of membrane-associated A␤ fibrillogenesis and neurotoxicity (5, 6). The formation of GM1-bound A␤, which is thought to be a seed for the formation of toxic amyloid fiber, depends on the concentration of Ch in model membranes prepared from GM1/Ch/sphingomyelin (SM) (7). Additionally, oligomeric A␤ can promote the release of l...

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Surface pressure (pi)-, surface potential (DeltaV)-, dipole moment (mu( perpendicular))-area (A) isotherms and morphological behavior at the air-water interface were obtained for multicomponent monolayers of two different systems for dipalmitoylphosphatidylcholine (DPPC)/egg-phosphatidylglycerol (PG) (= 68:22, by weight)/Hel 13-5 and DPPC/palmitic acid (PA) (= 90:9, by weight)/Hel 13-5 (Hel 13-5 is a newly designed 18-mer amphiphilic alpha-helical peptide with 13 hydrophobic and 5 hydrophilic amino acid residues). The phase behavior of these model systems was investigated on a subsolution of 0.02 M tris(hydroxymethyl)aminomethane (Tris) buffer (pH 8.4) with 0.13 M NaCl at 298.2 K by employing the Wilhelmy method, the ionizing electrode method, and fluorescence microscopy. Especially, the present study focuses on the interfacial effect of the addition of Hel 13-5 on two binary systems, DPPC/egg-PG and DPPC/PA monolayers, as the substitute for pulmonary surfactant proteins, and on the respective roles of PG and PA for the monolayers in the three-component systems. Constant kink points ( approximately 42 mN m(-1)) clearly appear on the pi-A isotherms, independent of the compositions in the ternary systems, which corresponds to the Hel 13-5 collapse pressure similar to that of SP-B and SP-C as functions in multicomponent monolayers. This implies that Hel 13-5 is squeezed out of ternary monolayers above approximately 42 mN m(-1), resulting in two- to three-dimensional phase transformation. Furthermore, Langmuir isotherms clearly show that Hel 13-5 with egg-PG is squeezed out of the DPPC/egg-PG/Hel 13-5 system, whereas only Hel 13-5 is squeezed out of the DPPC/PA/Hel 13-5 system. Cyclic compression and expansion isotherms of these systems were carried out to confirm the spreading and respreading capacities. In addition, the interfacial behavior of the ternary mixtures has been analyzed by the additivity rule. Morphological examinations and comparisons have verified the interactions of Hel 13-5 with the representative miscible mixture (DPPC/PA system) by fluorescence microscopy. Consequently, distinct morphological variations corresponding to the squeeze-out behavior are observed as a fluorescent contrast recovery. Herein, a new mechanism of the refluorescent phenomenon is proposed by varying the surface composition of Hel 13-5.

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 proteinlipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic ␣-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.Cellular organelles like the Golgi apparatus and the endoplasmic reticulum adopt characteristic structures depending on their function (1, 2). For example, extensive membrane nanotubules, typically 50 -70 nm in diameter and up to several m in length, have been observed to form the Golgi complex, the trans Golgi network, and the connections between the Golgi stacks. The morphological engineering of these membranes involve complex interactions between proteins and lipids that are not yet understood (3, 4). However, there must be some fundamental machinery required to form such structures.We recently described the properties of a de novo-designed 18-mer peptide, Hel 13-5 (5, 6). This peptide can adopt an ideal ␣-helix having a 240°hydrophobic sector region (Fig. 1A). It forms a self-association state in buffer solution by adopting this amphipathic structure (70% ␣-helical structure by CD), and it binds to model-and biomembranes with high affinity. In the present study, we show that Hel 13-5 induces nanotubular structures, not only for PC liposomes, but also for various naturally occurring phospholipids. Most importantly, we demonstrate that 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 of the Golgi apparatus. MATERIALS AND METHODSReagents-Peptide was synthesized by the Fmoc 1 strategy based on the solid phase technique starting from Fmoc-PAL-PEG resin using a PerSeptive 9050 automatic peptide synthesizer described previously (5). The stock solutions of Hel peptides were prepared as follows: the powders were damped with a small amount of 30% acidic acid and then diluted in buffer (5 mM Tes/100 mM NaCl, pH 7.4). The peptide concentrations in the buffer solution were determined from the UV absorbance of Trp at 280 nm (⑀ ϭ 5500).Turbidity Measurement-A lipid solution in chloroform was filmed in a round bottom flask by drying in a stream of N 2 gas. The lipid film was hydrated with the Tes buffer by vortexing. The turbid liposome solution obtained was then dilu...

The following peptides were synthesized by classical methods in solution: Ac-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-6) and Ac-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-NHCH3 (F-7). The rates of hydrolysis of the Arg-Gly bond in these peptides by thrombin were measured, and the rate for the Phe-containing peptide F-6 was found to be much larger than that for F-7. Previous work [van Nispen, J. W., Hageman, T. C., & Scheraga, H. A. (1977) Arch. Biochem. Biophys. 182, 227] has demonstrated the importance of Phe-Leu at positions P9-P8 of the A alpha chain of fibrinogen for the thrombin-fibrinogen interaction. This work demonstrates that the presence of Leu (P8) alone is insufficient to account for the enhanced hydrolysis rates and that the presence of Phe (P9) is essential for normal action of thrombin on the A alpha chain of fibrinogen.