Structural and Functional Characterization of the Newly Designed Antimicrobial Peptide Crabrolin21

Abstract

(1) Background: antimicrobial resistance is becoming a dramatic problem for public health, and the design of new antimicrobial agents is an active research area. (2) Methods: based on our previous work, we designed an improved version of the crabrolin peptide and characterized its functional and structural properties with a wide range of techniques. (3) Results: the newly designed peptide, crabrolin21, is much more active than the previous ones and shows specific selectivity towards bacterial cells. (4) Conclusions: crabrolin21 shows interesting properties and deserves further studies.

Keywords: CD spectroscopy; NMR spectroscopy; antimicrobial resistance; membrane specificity.

Figure 1

Kinetics of CF release from vesicles after addition of crabrolin21. Kinetics of CF release after addition of crabrolin21 to POPE/POPG (7:3 molar ratio) or POPC/cholesterol vesicles (1:1 molar ratio) (black and gray lines, respectively). Liposome concentration: 50 μM. Crabrolin21 concentration: 1.5 μM. Measurements were performed at 298 K and pH 7.4. Standard errors over three measurements are reported.

Figure 2

Secondary structure of crabrolin21 in different systems. Far UV circular dichroism spectra of crabrolin21 in aqueous buffer solution (solid red line), methanol (green dashed line), TFE (orange dotted line), and in the presence of POPE/POPG (7:3 molar ratio) at 500 µM total lipid concentration in phosphate buffer (10 mM, pH 6.5) (blue short-dashed line). Crabrolin21 concentration: 11 μM. Measurements were performed at 298 K and pH 7.4.

Figure 3

Crabrolin21 binding to membranes. Crabrolin21/membrane association followed through the peptide CD signal at 197 nm for POPE/POPG (7:3 molar ratio) or POPC/cholesterol vesicles (1:1 molar ratio) (blue circles and light blue triangles, respectively). Crabrolin21 concentration: 11 μM. Measurements were performed at 298 K and pH 7.4.

Figure 4

RBC lysis assay. The assay is based on hemoglobin release; circles are the experimental data points, while the continuous line is a guide for the eye. Error bars indicate the interval of duplicate measurements.

Figure 5

Solution structure of crabrolin21 and comparison with previous crabrolin peptides. (A): NMR solution structure of crabrolin21 peptide in TFE. The family of 10 conformers was obtained by torsion angle dynamics by using the CYANA 3.98 software [28]. The side chains of charged amino acids (arginines and lysines) of the first conformer are shown on the right side, and the hydrophobic side chains are shown on the left side. (B): Helical wheels of crabrolin21, crabrolin WT, crabrolin Minus, and crabrolin Plus with the charged residues colored in blue. (C): sequence alignment of crabrolin peptides. Conserved residues are marked with an asterisk, and charged residues are circled. Helical wheels were obtained with DrawCoil [35].

Figure 6

Phosphorous NMR of lipopolysaccharide from E. coli in the presence of increasing amounts of crabrolin21. LPS from E.coli was titrated with increasing amounts of crabrolin21. Measurements were performed at 298 K and pH 6.5.