NMR Characterization of the Structural Effects of Charge Repulsion in an Antibiotic Peptide Model

Abstract

Due to the decreasing effectiveness of common antibiotics, there is an overwhelming need for new antibiotics. One promising avenue is peptide antibiotics, which are generally helical and cationic. We are investigating peptide antibiotic models composed of the hydrophobic dialkylated amino acid Aib (α-aminoisobutyric acid), which imparts a strong 310-helical bias due to steric hindrance at the α-carbon. Cationicity is achieved by insertion of lysine residues. Previous studies have shown that insertion of adjacent neutral monoalkylated amino acids into an Aib sequence creates a region of reduced steric hindrance, allowing hydrogen-bonding solvents to disrupt the hydrogen bond that spans the insertion region, ultimately creating a kink in the helix. Separation of the monoalkylated residues by one turn leaves the helix undistorted. However, some studies suggest that insertion of charged residues one turn apart may distort helical structure. We report here the 3D NMR structural characterization of a model antibiotic peptide composed primarily of Aib, with two lysine residues placed one turn apart. Spectra were obtained in DMSO-d6 solution. Backbone and sidechain 1H and 13C resonances were assigned using natural abundance HSQC and CO-selective HMBC spectra. 1-D temperature dependence of amide chemical shifts indicated a 310-helical conformation with all intrahelical hydrogen bonds intact. Homonuclear ROESY crosspeaks were used to obtain 46 distance constraints, which together with the temperature data were used to calculate the structure using Xplor-NIH. The generated structure is fully 310-helical with a slight bend away from the charged face, likely due to charge repulsion. Thus, placement of charged amino acids has a small but noticeable effect on the structures of helical peptides even in a strongly hydrogen-bonding solvent. Our results indicate that the placement of cationic residues should be considered carefully in antibiotic peptide design.