Capacity for increased surface area in the hydrophobic core of β-sheet peptide bilayer nanoribbons

Abstract

Amphipathic peptides with amino acids arranged in alternating patterns of hydrophobic and hydrophilic residues efficiently self-assemble into β-sheet bilayer nanoribbons. Hydrophobic side chain functionality is effectively buried in the interior of the putative bilayer of these nanoribbons. This study investigates consequences on self-assembly of increasing the surface area of aromatic side chain groups that reside in the hydrophobic core of nanoribbons derived from Ac-(XKXE)₂ -NH₂ peptides (X = hydrophobic residue). A series of Ac-(XKXE)₂ -NH₂ peptides incorporating aromatic amino acids of increasing molecular volume and steric profile (X = phenylalanine [Phe], homophenylalanine [Hph], tryptophan [Trp], 1-naphthylalanine [1-Nal], 2-naphthylalanine [2-Nal], or biphenylalanine [Bip]) were assessed to determine substitution effects on self-assembly propensity and on morphology of the resulting nanoribbon structures. Additional studies were conducted to determine the effects of incorporating amino acids of differing steric profile in the hydrophobic core (Ac-X₁ KFEFKFE-NH₂ and Ac-(X_1,5 KFE)-NH₂ peptides, X = Trp or Bip). Spectroscopic analysis by circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy indicated β-sheet formation for all variants. Self-assembly rate increased with peptide hydrophobicity; increased molecular volume of the hydrophobic side chain groups did not appear to induce kinetic penalties on self-assembly rates. Transmission electron microscopy (TEM) imaging indicated variation in fibril morphology as a function of amino acid in the X positions. This study confirms that hydrophobicity of amphipathic Ac-(XKXE)₂ -NH₂ peptides correlates to self-assembly propensity and that the hydrophobic core of the resulting nanoribbon bilayers has a significant capacity to accommodate sterically demanding functional groups. These findings provide insight that may be used to guide the exploitation of self-assembled amphipathic peptides as functional biomaterials.

Keywords: amphipathic; nanoribbons; non-natural amino acids; self-assembling peptides; β-sheet fibrils.

FIGURE 1

Schematic illustrating self‐assembly of amphipathic Ac‐(XKXE)₂‐NH₂ peptides into β‐sheet bilayer fibrils. The hydrophobic side chains (X, green) are buried in the interior of the β‐sheet bilayer with the hydrophilic side chain (Lys and Glu) exposed to the aqueous environment. When X = Phe, the distance within the bilayer is 10 Å whereas 4.7 Å between β‐strands. The β‐sheet bilayer axis is perpendicular to the page

FIGURE 2

Structures of amino acids incorporated in the X position of Ac‐(XKXE)₂‐NH₂ variant peptides: phenylalanine (Phe), homophenylalanine (Hph), tryptophan (Trp), 1‐naphthylalanine (1‐Nal), 2‐naphthylalanine (2‐Nal), and biphenylalanine (Bip). Relative hydrophobicity is indicated in the form of partition coefficients (π) between octanol and water relative to glycine (defined as having a partition coefficient of 0 on this scale). Higher π values correspond to higher hydrophobicity. The calculated van der Waals volume for each amino acid is also shown in order to indicate the degree of change in steric profile for each amino acid

FIGURE 3

Proposed packing mode for antiparallel out‐of‐register amphipathic Ac‐X₁KFEFKFE‐NH₂ and Ac‐ (X_1,5KFE)₂‐NH₂ peptides with tryptophan (Trp) and biphenylalanine (Bip) substitutions at position 1 (X₁) or at positions 1 and 5 (X₅). As shown schematically in this diagram, the position 1 substituents are unpaired cross‐strand whereas the position 5 substituents are self‐paired

FIGURE 4

Circular dichroism spectra in 5% hexafluoroisopropanol/water at time periods of 0, 1, 2, 4, 12, and 24 h for amphipathic Ac‐(XKXE)₂‐NH₂ peptides in which X is (A) phenylalanine; (B) homophenylalanine; (C) tryptophan; (D) 1‐naphthylalanine; (E) 2‐naphthylalanine; and (F) biphenylalanine

FIGURE 5

Fourier transform infrared spectra for Ac‐(XKXE)₂‐NH₂ peptides in which X = phenylalanine, homophenylalanine, tryptophan, 1‐naphthylalanine, 2‐naphthylalanine, or biphenylalanine. Each peptide exhibits a characteristic amide I signal at ~1618 cm⁻¹ indicating β‐sheet secondary structure for all variants

FIGURE 6

Transmission electron microscopy images of Ac‐(XKXE)₂‐NH₂ peptide nanoribbons. (A) X = phenylalanine; (B) X = homophenylalanine; (C) X = tryptophan; (D) X = 1‐naphthylalanine; (E) X = 2‐naphthylalanine; and (F) X = biphenylalanine

FIGURE 7

(A) Circular dichroism (CD) spectra of Ac‐WKFEFKFE‐NH₂ in 5% hexafluoroisopropanol (HFIP)/water over 24 h; (B) CD spectra for Ac‐(WKFE)₂‐NH₂ in 5% HFIP/water over 24 h; and (C) Fourier transform infrared spectra for Ac‐BipKFEFKFE‐NH₂ and Ac‐(BipKFE)₂‐NH₂ (24 h, 5% d ₂‐HFIP/D₂O)

FIGURE 8

Circular dichroism (CD) and Fourier transform infrared (FT‐IR) spectra for the mono‐ and di‐substituted biphenylalanine (Bip) variants (0.2‐mM peptide). (A) CD spectra of Ac‐BipKFEFKFE‐NH₂ in 5% hexafluoroisopropanol (HFIP)/water over 24 h; (B) CD spectra for Ac‐(BipKFE)₂‐NH₂ in 5% HFIP/water over 24 h; and (C) FT‐IR spectra for Ac‐BipKFEFKFE‐NH₂ and Ac‐(BipKFE)₂‐NH₂ (24 h, 5% d ₂‐HFIP/D₂O)

FIGURE 9

Transmission electron microscopy images for the mono‐ and di‐substituted assembled materials. (A) Ac‐WKFEFKFE‐NH₂; (B) Ac‐(WKFE₂)‐NH₂; (C) Ac‐BipKFEFKFE‐NH₂; and (D) Ac‐(BipKFE₂)‐NH₂