Haruhiko Aoyagi

Relationship between antimicrobial activity and amphiphilic property of basic model peptides

Several cationic model peptides of the prepiece moieties of mitochondrial protein precursors were found to be active against Gram-positive bacteria, but inactive against Gram-negative bacteria. The CD spectra of the model peptides in the presence of phospholipid liposomes demonstrated that antimicrobial activity was generally in parallel with the content of the alpha-helical amphiphilicity. The results indicate that appropriate positioning of cationic and hydrophobic groups in the stable alpha-helical structure of the peptides is important to exhibit antimicrobial activity. These peptides also have an ability to leak carboxyfluorescein from acidic and neutral phospholipid vesicles, suggesting that the peptides interact with the bacterial membrane to perturb it.

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.

Effect of salts on conformational change of basic amphipathic peptides from β-structure to α-helix in the presence of phospholipid liposomes and their channel-forming ability

Abstract A synthetic model peptide, H-(Leu-Alα-Arg-Leu) 3 -(Leu-Arg-Alα-Leu) 3 -OH (4 6 ) can form ion channels in planar lipid bilayers by taking an amphipathic α-helix (Agawa, Y., Lee, S., Ono, S., Aoyagi H., Ohno, M., Taniguchi, T., Anzai, K. and Kirino, Y. (1991) J. Biol. Chem. 266, 20218–20222). For further study of ion channels formed by this type of peptides, we planned to synthesize [Trp 1 ]-4 6 (Ser) and [Trp 12 ]-4 6 (Ser) in which a hydrophilic amino acid, Ser, was introduced in several positions of 4 6 instead of hydrophobic ones. This modification was expected to decrease the ability of membrane perturbation and to simplify various current levels of the channel observed for 4 6 . Furthermore, additional Trp was introduced to the N-terminus or position 12 to monitor the lipid—peptide interaction. CD study showed that both peptides formed a random structure in buffer, but an α-helix in the presence of egg PC and a β-structure in egg PC/egg PG (3:1). Moreover, addition of NaCl to the acidic liposomes induced the conformational transition in the peptide from β-structure to α-helix. Salt-induced conformational transition in the presence of acidic liposomes was discussed in terms of membrane binding and ion-channel formation in planar lipid bilayer. Despite introduction of hydrophilic residues instead of hydrophobic residues in 4 6 , the peptide showed nearly the same dye-release ability from egg PC- egg PG liposomes as 4 6 . [Trp 12 ]-4 6 (Ser) was able to form cation-selective ion channels with two levels of conductance (mainly 250 and occasionally 125 pS) in asolectin planar lipid bilayer, suggesting that appropriate orientation of hydrophobic and hydrophilic residues in amphipathic peptide can simplify channel current levels.

Conformational studies of amphipathic α-helical peptides containing an amino acid with a long alkyl chain and their anchoring to lipid bilayer liposomes

In order to investigate the conformation and orientation of lipid-bound peptides and proteins in the lipid bilayer, basic amphipathic alpha-helical peptides with a long alkyl chain, palmitoyl-(Leu-Ala-Arg-Leu)3-NHCH3 (P-4(3)) and Ac-Leu-Ala-Arg-Leu-Trp-Amy-Arg-Leu-Leu-Ala-Arg-Leu-NHCH3 (Amy-4(3), Amy; alpha-aminomyristic acid) were designed and synthesized. The conformational features and spectroscopic behavior in a buffer solution and in neutral and acidic liposomes were studied by CD, dye-leakage, and fluorescence measurements. The CD data indicated that P-4(3) took an alpha-helical structure in aqueous solution and in neutral and acidic liposomes. On the other hand, Amy-4(3) took a beta-structure in aqueous solution and an alpha-helical structure in neutral and acidic liposomes. The conformational change of Amy-4(3) was confirmed by fluorescence study on lipid titration of the peptide. The dye-leakage experiment showed that both peptides interacted with acidic liposomes to perturb them, but less effectively than Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3)) which has no long alkyl group. Based on these results, a discussion is made concerning the conformation and orientation of peptides in aqueous solution and in the lipid bilayer.

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer.

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.

Synthesis of a peptide with cobrotoxin activity

Abstract A peptide with the sequence of cobrotoxin was prepared by Merrifields solid-phase method. The peptide obtained was found to show similar behavior to natural cobrotoxin in an immunodiffusion experiment and to effect the response to acetylcholine of frog muscle. Further purification of this peptide raised the toxic activity up to about 20% of that of cobrotoxin.

Syntheses of antibacterial peptides, gramicidin S analogs and designed amphiphilic oligopeptides

Abstract In order to clarify the relationship between antimicrobial activity and peptide-structure, gramicidin S analogs and cationic α-helical model peptides were designed and synthesized. Introduction of cationic side chains in hydrophilic side of gramicidin S increased antimicrobial activity against Gram-negative bacteria. Amphiphilic structures of the α-helical peptides were found to be effective to show antimicrobial activities against Gram positive bacteria. Increase in number of cationic amino acid residues in the α-helical peptides caused appreciable antimicrobial activities against Gram-negative bacteria, however, induced lower activities against Gram-positive ones.

δ and μ opiate receptor probes: fluorescent enkephalins with high receptor affinity and specificity

The fluorescent amino acid, L‐1‐pyrenylalanine (Pya) was incorporated into [D‐Ala2,Leu5]enkephalin and its methyl ester at position 4 or 5. Pya‐enkephalins showed strong fluorescent intensity and displayed high binding affinity for opiate receptors. Pya4‐enkephalins showed high specificity for the μ receptors, while Pya5‐enkephalins showed high specificity and selectivity for the δ receptors. Particularly, [D‐Ala2,Pya5]enkephalin was as potent as the most utilized δ‐specific ligand of [D‐Ala2,D‐Leu5]enkephalin (DADLE), and yet its δ‐selectivity was about 5‐times greater than that of DADLE. Thus, Pya‐enkephalins per se can be utilized as a fluorescent probe or tracer for the opiate receptor‐binding assays.

Efficient asymmetric synthesis of α-amino acids through hydrogenation of α,β-dehydroamino acid residue in cyclic dipeptides

Abstract A series of cyclo (Δaminoacyl-L-Ala) ( 4 ) (Δ=α,β-dehydro) were prepared from cyclo(Gly-L-Ala) and corresponding aldehyde, and hydrogenated with Pd black in MeOH. Chiral inductions producing cyclo (L-aminoacyl-L-Ala) ( 5 ) from 4 were 96–99% in the case of L-Aba (2-aminobutanoic acid), L-Val, L-Leu, and L-App (2-amino-5-phenylpentanoic acid) as an L-aminoacyl moiety in 5 . Pure L-Leu, L-Aba, and L-App were synthesized in preparative scale from corresponding 4 through asymmetric hydrogenation and acid-hydrolysis.

Environment-dependent conformation and antimicrobial activity of a gramicidin S analog containing leucine and lysine residues

An analog of gramicidin S, cyclo(‐L‐Leu‐L‐Lys‐L‐Leu‐D‐Leu‐L‐Leu‐)2, in which four out of five amino acid components of gramicidin S were substituted, has been synthesized. This analog assumes a conformation similar to that of gramicidin S in acidic liposomes and a random conformation in neutral liposomes. The antimicrobial activity of this analog corresponded to one‐fourth of that of gramicidin S. A possible mechanism for conformational changes in acidic liposomes is discussed.