Study On The Molecular Characteristics And Antimicrobial Activity Of Buckwheat Antimicrobial Peptide-FtAMP

Antimicrobial peptides(AMPs)are a series of small molecule peptides secreted by the innate immune system of organisms and play an important role in the inherent defense mechanisms of most organisms.Due to their broad-spectrum antimicrobial activity and unique membrane-action mechanism,AMPs are considered to be the best candidates as new anti-infectives to replace or accompany conventional antibiotics.However,its potential cytotoxicity,high manufacturing costs and weak physiological stability have been challenges for the further application of natural peptides.Therefore,in recent years,most researchers have performed molecular design changes on the amino acid sequence of natural AMPs in order to find key conserved sequences which affect their activity,in the hope of maximizing the antimicrobial activity and availability of AMPs.In this study,we use the prokaryotic expression system to obtain a Tartary Buckwheat FtAMP with trypsin-inhibiting activity.The predicted inhibitory site is 21-position arginine(Arg),and FtAMP-R21 A and FtAMP-R21 F mutants are obtained by site directed mutagenesis.The physicochemical properties,inhibitory and antimicrobial activities of the three peptides were compared in order to explore the relationship between the inhibitory and antimicrobial activity of FtAMP.Subsequently,we synthesized the ?-helix peptide(FtAMP-N,FtAMP-C)at the N/C-terminus of FtAMP molecule,and explored which helix of two alpha helices is at play.Then using FtAMP as a template,and selecting the ?-helical region with the most development space for antimicrobial activity.According to principle of helical wheel projection and substitution of specific amino acid residues,and around the structure of charge and amphiphilic molecules and try to molecular modification of it.Through the comparison of structure-function and activities of peptides,the structure-function relationship of FtAMP was explored.Our results indicated that the tartary buckwheat antimicrobial peptide(FtAMP)with trypsin inhibitory activity was expressed in Escherichia coli and purified.The purity reached above 95% and a yield of about 0.55 mg/L.SDS-PAGE analysis and MALDI-TOF MS indicated that the molecular weight was 5316.7 Da.Protease inhibitory activity assay revealed that FtAMP-R21 A and FtAMP-R21 F essentially lost the inhibitory activity on trypsin,but they inhibited elastase and ?-chymotrypsin respectively,two new protease inhibitors were obtained.It is suggested that Arg-21 is a specific site for the trypsin inhibitory activity of FtAMP.Antimicrobial test showed that the peptides all have significant inhibitory effect against Trichoderma koningii,Rhizopus chinonsis,and Fusarium oxysporum,but they do not seem to affect the growth of bacteria.These results indicate that changes in the protease inhibitory activity of the FtAMP have no affect its antifungal activity.Therefore,the antimicrobial activity of FtAMP is attributed to ?-helical structure in its molecule.Experiments have shown that both FtAMP-N and FtAMP-C have good antibacterial activity.Subsequently,a series of FtAMP mutants(FtAMP-E12 A,FtAMP-E12A/E9 K,FtAMP-E12A/E9 A,and FtAMP-E12A/E9K/T24E)were expressed based on the helical wheel projection method.Circular dichroism analysis showed that the secondary structures of the 4 peptides were folded into ?-helical structure with typical double negative peaks at 208 nm and 222 nm,and indicating that the amino acid mutation and protein expression did not change the spatial structure of the parent peptide.Antifungal analysis showed that inhibitory effect of the mutants on the fungus was enhanced observably stronger and FtAMP.In particular,the effectiveness of FtAMP-E12A/E9 K was improved approximately 2-fold compared to FtAMP.And its hemolytic activity was observably lower than FtAMP,the selectivity indexes of FtAMP-E12A/E9 K against fungi were improved approximately 3-fold compared to FtAMP,it is hopeful to become an effective antibacterial agent.This study provides theories foundation for the further study of the molecular mechanism and function of antimicrobial peptides.