Non-perfectly Amphipathic α-Helical Structure Containing the XXYXX Sequence Improves the Biological Activity of Bovine α_s2-Casein Antimicrobial Peptides

Abstract

Non-amphiphilic WIQPKTKVIPYVRYL (WI-6) derived from bovine α s2 -casein f (193−207) was modified by a defined mutation method to obtain five engineered peptides with mirror symmetry structures. The five engineered peptide sequences were WF-1 (WFQVKTRVRTKVQFW), FW-2 (FWRRYKKVKKYRRWF), FW-3 (FWQVIKKVKKIVQWF), FK-4 (FKQFYRRVRRYFQKF), and FR-5 (FRQWYRRVRRYWQRF). However, FW-2, FW-3, FK-4, and FR-5 had obvious XXYXX sequences. Among these, FW-3 was demonstrated to have the highest antibacterial activity, which indicates that the non-perfectly amphipathic α-helical structure containing the XXYXX sequence has a better bactericidal effect. Therefore, peptide FW-3 could be widely used as a substitute for antibiotics in food, medicine, and other fields. These findings provide a potential method for designing novel antimicrobial peptides.

Antimicrobial Peptides with Rigid Linkers against Gram-Negative Bacteria by Targeting Lipopolysaccharide

“…The cytotoxicity of all peptides toward HEK-293 cells was evaluated using MTT assay. Figure A shows that at the highest concentration tested (512 μM), the cell viabilities were 71 and 64% after treatment with peptides Lf(28–34), and Ce(1–8), respectively, while peptides CL3 and CL4 exhibited higher cytotoxicity, with cell viability below 10%, probably due to the greater positive charges in CL3 (net charge: +9) and the higher hydrophobicity in CL4 ( H : 0.251) (Table ) leading to their enhanced electrostatic and hydrophobic interactions with mammalian cell membranes, respectively, ultimately resulting in their increased cytotoxicity. , HEK-293 cells maintained 60%–82% of cell viability after treatment with peptides CL1, CL2, and CL5–CL7 at 512 μM. In particular, CL5 had less effect on HEK-293 cells, with a cell viability of 97% at 256 μM and 82% at 512 μM.…”

“…The minimum inhibitory concentrations (MICs) of the peptides against bacteria were determined by the broth microdilution method . The bacteria cultured to the logarithmic phase in Luria–Bertani (LB) broth were diluted to 1–5 × 10 5 cfu/mL.…”

“…The minimum inhibitory concentrations (MICs) of the peptides against bacteria were determined by the broth microdilution method. 33 The bacteria cultured to the logarithmic phase in Luria−Bertani (LB) broth were diluted to 1−5 × 10 5 cfu/mL. The peptides were dissolved in sterile deionized water to a concentration of 512 μM and then 2-fold serially diluted in a 96-well plate.…”

In Silico Development of Novel Chimeric Lysins with Highly Specific Inhibition against Salmonella by Computer-Aided Design

“…LeuA is a bacteriocin from Leuconostoc mesenteroides, which is characterized by a narrow spectrum and low antibacterial activity (Figure A) . The α-helix Trp18-Gly35 of LeuA was replaced with amino acids to enhance its charge and hydrophobicity to improve its antibacterial activity (Figure B) . Based on the data set from the collection of antimicrobial peptides (CAMP, Biomedical Informatics Centre, National Institute for Research in Reproductive Health), we used Support Vector Machine (SVM), Random Forest, and Discriminant Analysis algorithms to analyze the possibility of the designed peptide (LeuA-P) as an antimicrobial peptide .…”

“…18 The α-helix Trp18-Gly35 of LeuA was replaced with amino acids to enhance its charge and hydrophobicity to improve its antibacterial activity (Figure 1B). 19 Based on the data set from the collection of antimicrobial peptides (CAMP, Biomedical Informatics Centre, National Institute for Research in Reproductive Health), we used Support Vector Machine (SVM), Random Forest, and Discriminant Analysis algorithms to analyze the possibility of the designed peptide (LeuA-P) as an antimicrobial peptide. 20 LeuA-P was synthesized by Genscript (Nanjing, China), and its minimum inhibitory concentration (MIC) was determined.…”