Substitution of tyrosine with electron-deficient aromatic amino acids improves Ac-PHF6 self-assembly and hydrogelation

Abstract

The hexapeptide PHF6 (VQIVYK), an amyloidogenic peptide stretch from human tau, self-assembles via parallel in-register β-sheet formation, wherein Tyr residues are involved in aromatic stacking interactions. Ac-PHF6 (CH₃CO-VQIVYK-NH₂) forms a viscous solution in water but causes instant gelation of PBS and cell culture media. Aromatic substitutions have been reported in the literature to modulate the self-assembly of peptides. In this study, we perturbed the electronic properties of the sole aromatic residue in Ac-PHF6 and studied hydrogelation. The Tyr residue was substituted with Phe, and the phenyl moiety was then substituted with various electron-withdrawing groups at the para position. All peptides caused PBS gelation with comparable rheological properties. The structures underlying the hydrogels were β-sheet fibrils. The electron-deficient aromatic moieties improved self-assembly and hydrogelation. Ac-PHF6 and no other aromatic analog except the one having p-(trifluoromethyl)phenylalanine caused the gelation of deionized water. Water gelation caused by the p-(trifluoromethyl)phenylalanine-containing analog is likely hydrophobicity-driven.

Fig. 1. Electrostatic charge density maps of toluene (A), p-cresol (B), 4-fluorotoluene (C), 4-cyanotoluene (D), 4-nitrotoluene (E), and 4-(trifluoromethyl)toluene (F).

Fig. 2. Inverted vials of Ac-PHF6 and its analogs showing PBS gelation. (A) Ac-PHF6, (B) Ac-VQIVFK-am, (C) Ac-VQIVF(fl)K-am, (D) Ac-VQIVF(CN)K-am, (E) Ac-VQIVF(NO₂)K-am, and (F) Ac-VQIVF(CF₃)K-am.

Fig. 3. Rheology of PBS gels. Plot of G′ and G′′ against angular frequency for 20 mM gels of (A) Ac-PHF6, (B) Ac-VQIVFK-am, (C) Ac-VQIVF(fl)K-am, (D) Ac-VQIVF(CN)K-am, (E) Ac-VQIVF(NO₂)K-am, and (F) Ac-VQIVF(CF₃)K-am.

Fig. 4. CD spectra of Ac-PHF6 analogs, (A) Ac-PHF6, (B) Ac-VQIVFK-am, (C) Ac-VQIVF(fl)K-am, (D) Ac-VQIVF(CN)K-am, (E) Ac-VQIVF(NO₂)K-am, and (F) Ac-VQIVF(CF₃)K-am.

Fig. 5. ATR-FTIR spectra of Ac-PHF6 and its analogs. The red traces are the spectra recorded for water samples, while the black traces are the spectra recorded for PBS samples. (A and B) Ac-PHF6, (C and D) Ac-VQIVFK-am, (E and F) Ac-VQIVF(fl)K-am, (G and H) Ac-VQIVF(CN)K-am, (I and J) Ac-VQIVF(NO₂)K-am, and (K and L) Ac-VQIVF(CF₃)K-am.

Fig. 6. ThT fluorescence spectra of Ac-PHF6 and its analogs. (A) Ac-PHF6, (B) Ac-VQIVFK-am, (C) Ac-VQIVF(fl)K-am, (D) Ac-VQIVF(CN)K-am, (E) Ac-VQIVF(NO₂)K-am, and (F) Ac-VQIVF(CF₃)K-am.

Fig. 7. MD simulations of the peptide steric zippers. (A) The VQIVYK steric zipper obtained from WALTZ-DB database. The middle structure of the largest cluster for (B) Ac-PHF6, (C) Ac-VQIVFK-am, (D) Ac-VQIVF(fl)K-am, (E) Ac-VQIVF(CN)K-am, (F) Ac-VQIVF(NO₂)K-am, and (G) Ac-VQIVF(CF₃)K-am. (H) The RMSD plots obtained from the trajectories of simulations.

Fig. 8. TEM images showing the structures underlying the PBS gels. (A) Ac-PHF6, (B) Ac-VQIVFK-am, (C) Ac-VQIVF(fl)K-am, (D) Ac-VQIVF(CN)K-am, (E) Ac-VQIVF(NO₂)K-am, and (F) Ac-VQIVF(CF₃)K-am. The scale bars represent 200 nm.