Effect Of Basic Amino Acid And Coiled Coil On Biological Activity Of Membrane Active Peptide HPRP-A1

It was reported that HPRP-A1 is a ?-helical cationic antibacterial peptide with good antibacterial and anticancer activity.Based on previous research,in this study,we focus on the relationships between the substitution of basic amino acids and coiled-coil structure with the biological activity of HPRP-A1,and this project divided two parts as follow.Part 1,since most membrane active peptide rich in positive charge and arginine(R)?Lysine(K)are the two most important basic amino acids.So,in this part,arginine was used to substitute Lysine in HPRP-A1 with different numbers and to explore the influence of basic amino acid to the biological activity of membrane activated peptide.Firstly,the hydrophobicity and self-aggregation ability of peptide were measured by reversed-phase high performance liquid chromatography(RP-HPLC).The results confirmed that the number of arginine positively correlated to the tendency of hydrophobicity,and negatively correlated to the self-aggregation ability of peptide;Secondly,antibacterial test,MTT test and hemolysis test were performed to explore the relationships between the biological activities and numbers of basic amino acid substitution.The data showed that the increasing of arginine enhanced the anticancer activity of AMPs against the He La cells,while antibacterial activity was reduced and the hemolytic activity was enhanced at the same time.Finally,NPN and ONPG data indicated that the penetrating ability of peptide against bacterial inner and outer membrane was weakened with the increasing numbers of arginine.Tryptophan fluorescence quenching experiment was also used to explore the interaction between peptides and mimic phospholipid membrane and got the same results.The amino acid substitution kept the total number of positive charge,while the hydrophobicity and helicity of the peptide was enhanced with the increasing of arginine content,the strong binding ability resulted to the decreasing of insertion depth of peptide,then its penetration ability decreased causing the decreasing of antibacterial activity.Comparing to primary amine of lysine,the guanidine of arginine decreases the penetrability of peptide against the cell membrane.These results are consistent with the membrane permeability experiment,which further confirmed that the antibacterial activity of AMPs is closely related to its self-aggregation ability.Part 2,part ?-helical peptides have seven repeated amino acid sequences called coiled coil,A and D positions are generally filled by hydrophobic amino acids,which could form a nonpolar surface,E and G positions tend to be occupied by a charged amino acid,which could create charge pairs,opposite charge via electrostatic interactions contribute to the stability of the coiled coil.In contrast,the same charge could break its stability.In order to explore the influence of coiled coil on the activity of HPRP-A1,we replaced the amino acid at A or D position in coiled coil,while the replacement did not change the amino acid component of peptide sequence.Firstly,the hydrophobicity of peptide was determined by RP-HPLC,antibacterial test,MTT test and hemolysis test were utilized to explore the effect of substitution on biological activity.The results indicated that the stability of coiled coil and hydrophobicity of AMP was positively correlated to biological activity.Furthermore,the anticancer mechanism of AMP was evaluated via LDH assay and apoptosis test.The data confirmed that HPRP-A1 and its analogues entered into HeLa cells by disrupting the cell membrane rather than endocytosis.In addition,they could induce the cancer cell apoptosis by mitochondria pathway;Finally,NPN and ONPG test was used to determine penetrating ability of peptide against the bacterial inner and outer membrane.It suggested that peptide enable to destruct the bacterial cell membrane.Liposome experiments further illustrated the damage of phospholipid membranes caused by peptide.The trends are consistent with its biological activities.The hydrophobicity of HPRP-A1-L6K7 was significantly reduced due to the lack of coiled coil,which led to reduce the damage ability against the mimic membrane.The leucine in A and D of peptide HPRP-A1 and HPRP-A1-L15K7 formed two leucine zipper structure,resulted to the strong membrane damage ability.The membrane broken ability of HPRP-A1-K8L15 decreased due to the lower hydrophobicity.These results further illustrated that the bioactivities of AMPs were closely related to the coiled coil structure and the hydrophobicity.