Effect of Introducing Leucine Residues into 2‑Aminoisobutyric Acid-Based Amphiphilic Helical Peptides on Intermolecular Interactions and Peptide Self-Assembly

Abstract

In recent years, self-assembling de novo-designed peptides have attracted increasing attention as materials for constructing submicrometer-scale structures. We have previously conducted structure-activity relationship studies on the BXBA-20 series of 20-residue amphiphilic peptides containing 2-aminoisobutyric acid (Aib) [Higashimoto, Y. et al. (1999) J. Biochem. 125, 705-712; Hara, T. et al. (2001) J. Biochem. 130, 749-755; Taira, J. et al. (2010) J. Pept. Sci. 16, 607-612]. These peptides share the common sequence Ac-(Aib-Xxx-Aib-Ala)₅-NH₂, where the introduction of a hydrophilic amino acid at the Xxx position results in the formation of a hydrophilic face on one side of the helix. Among them, some peptides exhibited weak intermolecular association and interactions with lipid membranes, though none demonstrated the ability to form submicrometer-scale structures through self-assembly. In this study, we present BKBL-20, a new member of this peptide series incorporating leucine on the hydrophobic face and lysine on the hydrophilic face. Circular dichroism spectroscopy indicated strong association in buffer, while hemolysis assays revealed significant membrane disruption. Transmission electron microscopy further confirmed the formation of ordered fibrous or array-like structures. These findings suggest that BKBL-20 successfully combines self-assembly and membrane-disruptive functions.

1

Structures of the model peptides evaluated in this study. The left panel shows the name and sequence of the model peptides. The center panel shows the helical wheel (left) and helical net (right) diagrams of BKBL-20. 2-aminoisobutyric acid is denoted as Aib or B. The three-dimensional structure of BKBL-20 was predicted using PEP-FOLD3, in which Aib residues in the peptide sequence were substituted with Val for modeling purposes. Cationic lysine and hydrophobic leucine residues are colored red and blue, respectively.

2

CD spectra of the monomeric model peptides: BKBL-20/16/12 and BKBA-20. (A) The CD spectra collected in 50 mM phosphate buffer (KPi, pH 7.4) and 2,2,2-trifluoroethanol are shown as black and red lines, respectively. (B) Concentration-dependent changes in the CD spectra of the model peptides. All measurements were conducted in 50 mM KPi (pH 7.4).

3

Structure, interactions, and perturbation activity of monomeric peptides in lipid bilayers. (A) The CD spectra of monomeric peptides (BKBA-20, BKBL-20, BKBL-16, and BKBL-12) measured in 50 mM KPi (pH 7.4) and in the presence of 1 mM DPPC SUVs are shown as black and red lines, respectively. The peptide concentration is 10 μM (Peptide/Lipid ratio, 1:100). (B) Hemolytic activity of monomeric peptides against human erythrocytes. The peptide concentrations used were 10, 5, and 1 μg/mL. Hemolytic activity was assessed relative to hemolysis by 1% Triton X-100 treatment.

4

Comparison of the CD spectra of the dimeric model peptides d-BKBL-16 and d-BKBL-12. (A) Concentration dependence of d-BKBL-16 and d-BKBL-12 in 50 mM KPi (pH 7.4). The CD spectra were measured at two peptide concentrations: 10 and 100 μM. (B) CD spectra of d-BKBL-12 and d-BKBL-16 in 50 mM KPi (black) and TFE (red). Peptide concentrations were 10 μM.

5

TEM images of BKBL-20. On the left is the image acquired at a magnification of 1100×. On the right is the image acquired at a magnification of 2700×. The right image shows an enlarged view of the region outlined by the black border in the left image.