Antimicrobial peptides (AMPs) have gained fast and growing interest in the past decades as they represent a viable alternative for the increasing bacterial resistance to conventional antibiotics. The cationic charge and amphipathicity of AMPs play an important role in their antimicrobial activity. The antimicrobial mechanism is still under debate, although a few models have been proposed to date, including barrel-stave, carpet, and toroidal-pore models. The secondary structure of cationic AMPs could highly affect the antimicrobial activity, which implies it has a significant role in the AMPs mechanism of action. Some artificially-designed AMPs can also form supramolecular assemblies. However, the specific and interconnected roles of the secondary structure and/or the self-assembled structures of AMPs on their antimicrobial activity and mechanism of action are not fully understood. Here, we explore the effect of pH and time on the secondary structure and the supramolecular assemblies of a well-studied AMP, GL13K (GKIIKLKASLKLL-NH2). GL13K was derived from a potential host defense salivary protein BPIFA2, and it is bactericidal to Gram-negative and Gram-positive bacteria and their biofilms.
GL13K and its randomized non-antimicrobial sequence, GL13K-R (IGIKLLKSKLKAL-NH2) were prepared in sodium borax buffers at increasing pH=8.0-10.6 to further investigate the effect of peptide charge on their structural changes. The calculated net charge of GL13K and GL13K-R is +5 at pH 7 and 0 at pH 14 (pI). Thus, the selected pH range has the highest change in net charge. The secondary structures of GL13K and GL13K-R were explored with circular dichroism (CD) spectroscopy at different times after peptide dissolution up to 9 days. To better understand the mechanism by which the secondary structures govern self-assembly of these peptides, the supramolecular structures were characterized by negative stained transmission electron microscopy (TEM) and dynamic light scattering (DLS).
The two peptides showed notable differences in structural conformations and assemblies as a function of both pH and time. Whereas the non-antimicrobial GL13K-R maintained unstructured configurations at all pH and times tested, the antimicrobial GL13K underwent a significant transformation to beta-sheet configurations at pH>9.6. These transformations occurred over time at pH=9.6-10.0, but at pH>10.0 stable beta-sheet configurations were detected right after peptide dissolution. TEM images showed that beta-sheet signal of GL13K at pH>9.6 was caused by the peptides self-assembly to twisted ribbons with thickness of 5-30 nm and length of as long as a few micrometers. DLS analysis confirmed the formation of these supramolecular assemblies. These results highlight the relevance of the secondary structure and supramolecular assemblies of the peptides formed as charge neutralization occur and how it might assist in the unraveling of the antimicrobial mechanism of these designed AMPs.