Determining the Charge State of Histidine Side Chains in Antimicrobial Piscidin By Nuclear Magnetic Resonance

Abstract

Piscidins constitute a family of three antimicrobial peptides discovered in the mast cells of hybrid striped bass. These peptides, which are highly cationic, contain several arginine and histidine residues. While piscidin 1 is the most antimicrobial and hemolytic isoform, piscidin 3, which has slightly lower antimicrobial activity, is significantly less hemolytic. One of the most striking differences between piscidin 1 and 3 is the substitution of glycine for the histidine at position 17 in piscidin 1.As part of its mechanism of action, piscidin recognizes negatively charged microbial membranes. Therefore, studying the interactions of the piscidin with lipids can help us better understand the chemical basis of its antimicrobial and hemolytic effects. Because physiological pH is around 7.4, and the average pKa of histidine side chains is around 6.0, a detailed study of the histidine side chains in piscidin 1 and 3 is needed to discern the charge state of the peptides under physiological conditions. In this research, we used solution nuclear magnetic resonance to obtain the pKa of the histidine side chains of piscidin bound to sodium dodecyl sulfate micelles. Heteronuclear multiple quantum coherence experiments were performed on piscidin 1 and 3 containing 15N-side chain labeled histidines. 15N and 1H chemical shifts were recorded as a function of pH to determine the titration curve of each histidine residue. The results will be discussed in the context of structure-function relationships in membrane-active peptides. The knowledge gained from these studies can help identify common principles that will facilitate the design of pharmaceuticals with broad-spectrum antibacterial activity, minimum induction of bacterial resistance, and low toxicity to mammalian cells.