Self-assembly dynamics and antimicrobial activity of all l- and d-amino acid enantiomers of a designer peptide

Abstract

Recent studies have shown that antimicrobial peptides (AMPs) can self-assemble into supramolecular structures, but this has been overlooked as causative of their antimicrobial activity. Also, the higher antimicrobial potency of d-enantiomers compared to l-enantiomers of AMPs cannot always be attributed to their different resistance to protease degradation. Here, we tested all l- and d-amino acid versions of GL13K, an AMP derived from a human protein, to study structural links between the AMP secondary structure, supramolecular self-assembly dynamics, and antimicrobial activity. pH dependence and the evolution of secondary structures were related to a self-assembly process with differences among these AMPs. The two GL13K enantiomers formed analogous self-assembled twisted nanoribbon structures, but d-GL13K initiated self-assembly faster and had notably higher antimicrobial potency than l-GL13K. A non-antimicrobial scrambled amino acid version of l-GL13K assembled at a much higher pH to form distinctively different self-assembled structures than l-GL13K. Our results support a functional relationship between the AMP self-assembly and their antimicrobial activity.

Fig. 1

(a-c) molecular structures and Mw of L-GL13K, D-GL13K, and L-GL13K-R. Cationic residues are marked in green and highly hydrophobic residues are marked in red. (d-f) titration curves and pKa values of L-GL13K, D-GL13K, and L-GL13K-R.

Fig. 2

CD spectra of 0.1 mM L-GL13K in borax-NaOH buffer with pH ranging from 9.4 to 10.8 for 0 day, 1 day, 2 days, 4 days, and 8 days.

Fig. 3

TEM images of negatively stained 0.1 mM L-GL13K agglomerations or self-assembled supramolecular structures (nanofibrils and nanoribbons) in buffer solutions of (a) pH 9.4 after 8 d, (b) pH 9.8 after 2 d, (c) pH 9.6 after 8 d, (d) pH 9.8 after 8 d, (e) pH 10.0 after 2 d. The inset plots are the corresponding CD spectra. (f) Cryo-TEM image of 1 mM L-GL13K nanoribbons in buffer solution at pH 10.8 for 1 d. The nanoribbons were embedded in vitreous ice and the arrow points at the lacey carbon/formvar support grid. All scale bars are 100 nm.

Fig. 4

Estimation of secondary structure (α-helix, β-sheet, β-turn, and unordered) contents from CD spectra of 0.1 mM L-GL13K in buffer solutions of pH 9.6, 9.8, and 10.0 for up to 8 days. Content values were averaged from estimations by three different methods, SELCON, CDSSTR, and CONTIN/LL.

Fig. 5

Inverted CD spectra of 0.1 mM D-GL13K in borax-NaOH buffer with pH ranging from 9.4 to 10.8 for 0 day, 1 day, 2 days, 4 days, and 8 days.

Fig. 6

Estimation of secondary structure (α-helix, β-sheet, β-turn, and unordered) contents from inverted CD spectra of 0.1 mM D-GL13K in buffer solutions of (a) pH 9.8 and (b) pH 10.0. Content values were averaged from estimations by three different methods, SELCON, CDSSTR, and CONTIN/LL. (c) TEM image of negatively stained D-GL13K self-assembled nanoribbons in pH 9.8 solution for 2 days. Inset is the corresponding inverted CD spectrum. Scale bar is 50 nm.

Fig. 7

(a-d) CD spectra of 0.1 mM L-GL13K-R in borax-NaOH buffer solutions with pH ranging from 9.0 to 12.0 for 0 day, 1 day, 2 days, 4 days, and 8 days. (e, f) TEM images of negatively stained L-GL13K-R aggregates and nanospheres in pH 11.0 and 12.0 buffer solutions, respectively for 1 day. Arrows point at small aggregates. All scale bars are 100 nm.