Systematic Moiety Variations of Ultrashort Peptides Produce Profound Effects on Self-Assembly, Nanostructure Formation, Hydrogelation, and Phase Transition

Abstract

Self-assembly of small biomolecules is a prevalent phenomenon that is increasingly being recognised to hold the key to building complex structures from simple monomeric units. Small peptides, in particular ultrashort peptides containing up to seven amino acids, for which our laboratory has found many biomedical applications, exhibit immense potential in this regard. For next-generation applications, more intricate control is required over the self-assembly processes. We seek to find out how subtle moiety variation of peptides can affect self-assembly and nanostructure formation. To this end, we have selected a library of 54 tripeptides, derived from systematic moiety variations from seven tripeptides. Our study reveals that subtle structural changes in the tripeptides can exert profound effects on self-assembly, nanostructure formation, hydrogelation, and even phase transition of peptide nanostructures. By comparing the X-ray crystal structures of two tripeptides, acetylated leucine-leucine-glutamic acid (Ac-LLE) and acetylated tyrosine-leucine-aspartic acid (Ac-YLD), we obtained valuable insights into the structural factors that can influence the formation of supramolecular peptide structures. We believe that our results have major implications on the understanding of the factors that affect peptide self-assembly. In addition, our findings can potentially assist current computational efforts to predict and design self-assembling peptide systems for diverse biomedical applications.

Figure 1

Illustration of the moiety variations carried out on a single amino acid residue at a time. 1: Me(β ↔ γ ↔ δ) shifts, 2) (CH₂ ↔ S) interconversion, 3) para-Ph(OH→H) conversion, 4) n(1 ↔ 2) interconversion, 5) β/γ(CO₂H → CONH₂) conversion, 6) α(CO₂H → CONH₂) conversion.

Figure 2

Depiction of the myriad changes in nanostructures that occurred when Ac-IVD was subjected to various moiety variation.

Figure 3

Scanning electron micrographs of Ac-ILE. (a) At 5 mg/mL, Ac-ILE dissolved completely and microbeads were formed; magnification 5000×. (b) At 20 mg/mL, Ac-ILE formed a hydrogel, with microbeads (yellow arrow), crystallites (blue arrow), and fibrils (dispersed throughout) being observed; magnification 1000×. (c) At 20 mg/mL, fusion of the beads into fibrils was observed; magnification 20000×. This suggests that the beads are precursors to fibril formation.

Figure 4

Scanning electron micrographs of Ac-LLE-NH₂: (a) Hydrogel at 25 mg/mL illustrates the biphasic equilibrium between microbeads and fibrils; magnification 5000×. (b) Supernatant at 40 mg/mL; magnification 2000×. (c) precipitate at 40 mg/mL; magnification 5000×. B and C show that the microbeads can also co-exist with the crystalline phase.

Figure 5

Scanning electron micrographs illustrating the four general morphologies observed with the acetylated tripeptides: (a) Amorphous structure (Ac-LVE, supernatant of 20 mg/mL; 5000×). (b) Bead microstructure (Ac-LLE, solution of 20 mg/mL; 5000×). (c) Crystalline nanostructure (Ac-LVE, precipitate of 20 mg/mL; 5000×). (d) Fibrillar nanostructure (Ac-IVD, hydrogel at 20 mg/mL; 10000×). (e) Illustration of a typical set-up to assess the self-assembly and aggregation of peptides. A series of peptide concentrations (Ac-LLE-NH₂ here) from 5–40 mg/mL, in steps of 5 mg/mL were prepared. This series also illustrates the four states generally observed in this study: solution (5–20 mg/mL), hydrogel (25 mg/mL; upturned vial), supernatant and precipitate (30–40 mg/mL).

Figure 6

X-ray crystal structure of Ac-LLE. Four blocks of Ac-LLE are shown and colored differently. (Left) hydrophobic interaction by the intercalating side chains of Leu1 and Leu2. (Right) hydrogen bond network. Intra- and inter-block hydrogen bonds are colored black, blue and magenta, respectively. The 2-fold axis relating the yellow and the green blocks are in green, and the 2-fold screw axis relating the yellow and the cyan blocks are in cyan.