| dc.description.abstract | Indolicidin (IL), a naturally synthesized peptide from bovine neutrophils, is a tryptophan-rich cationic antimicrobial peptide composing of only 13 amino acids. It is effective against a broad spectrum toward many kinds of pathogens. Therefore, it is regarded as a potential new-generation peptide antibiotic. However, similar to most antimicrobial peptides, indolicidin suffers from its possible disruption of erythrocytes. Recently, many researchers have paid much attention to design an IL-analogue with high antimicrobial activity but low hemolytic activity for applying in therapy, Systematically changing each amino acid in sequence has been tried to obtain an IL-analogue with high antimicrobial but low hemolytic activity. Limited success was obtained. A lot of efforts were then put into understanding the bactericidal and hemolytic mechanisms of indolicidin. Rational design can be taken after these mechanisms are understood. Many mechanisms have been proposed. Most of them were related to membrane kperturbation. We conjectured that IL peptide may perturb bacteria and erythrocyte cells with different extents arisen from the difference of the membrane compositions. We then try to evaluate the importance of each amino acid in sequence through the help of molecular dynamics simulation. Three IL analogues owning different antibacterial and hemolytic activities are designed by altering the important positions. Besides, it has been found that the aggregation of IL peptide in aqueous phase is related to its hemolysis. Furthermore, the fluorescence measurement and analysis were used to investigate whether the designed IL-analogues would form aggregates in aqueous solution or not and further investigated the actions of aggregative peptide-membrane interaction to evaluate the relationships between peptide structure and membrane perturbation. Criteria for peptide design can then be obtained.
The all-atom molecular dynamics simulation of interaction of IL peptide and two model lipid bilayers were performed with total simulation time up to 4??s to reveal the processes of IL adsorption onto and insertion into the membranes. The zwitterionic phospholipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), were used to mimic mammal cell membrane. The bacterial cell membrane was modeled by the mixture of POPC and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoglycerol (POPG). The packing order of lipid bilayer and the distribution of hydrophilic heads of phospholipids were presumably correlated to the membrane stability. It was found that the order of the mixed POPG/POPC lipid bilayer is reduced significantly upon the adsorption of IL. On the other hand, the order of the pure-POPC lipid bilayer is perturbed only slightly during the adsorption stage, but greatly reduced during the insertion stage. The results implied that the enhancement of IL adsorption on the microbial membrane may amplify its antimicrobial activity, while hemolysis may be reduced by the inhibition of IL insertion into the hydrophobic region of erythrocyte membrane. Three lower hemolytic IL-analogues including IL-K7 (Pro7 ? Lys), IL-F89 (Trp8 & Trp9 ? Phe) and IL-K7F89 (Pro7 ? Lys, Trp8 & Trp9 ? Phe) were further design from the analysis of critical amino acids in membrane perturbation. Moreover, the simulations of interaction between IL-F89 peptide and two model lipid bilayers were performed to discuss the relationships between membrane perturbation and biological activity. The results suggested that the perturbation of the hydrophobic core of lipid bilayer by single peptide is strongly related to its biological activities. Therefore, the molecular dynamics simulation of peptide-model membrane interaction successfully facilitated to design a peptide with higher antibacterial and at the same time lower cytotoxicity.
Moreover, the structure-activity relationships of indolicidin and its analogues were investigated through their interactions with the bacterium-like and erythrocyte-like model membranes. The intrinsic fluorescence and acrylamide quenching analysis were used to examine three designed IL-analogues would form aggregates in aqueous solution or not, and the results revealed that these peptides aggregate into different sizes in solution by hydrophobic interaction. The amounts of the peptide adsorption to model membrane, relative insertion depth of peptide in membrane, and degree of peptide aggregates dispersion in membrane, were defined to connect the peptide actions toward the model membranes to the bioactivities of the peptides. In particular, the roles of peptide aggregation to its bioactivities were interpreted. It was found that the reduction of peptide hydrophobicity reduces the peptide adsorption and insertion of peptides to the erythrocyte-like membrane. Subsequently, the hemolytic activity is reduced. On the other hand, the increase in peptide electropositivity would enhance the antibacterial activities. But the increase in electropositivity does not necessarily increase peptide-membrane binding. Instead, the peptide hydrophobicity facilitates aggregate formation in aqueous solution and the role of positive charges is to assist aggregate dissembling in the negatively charged bacterium-like membranes. Therefore, the peptide aggregation in solutions and dissembling in membranes play important roles in the antibacterial activity of indolicidin and its analogues.
By coupling the molecular dynamics simulations and experimental data analysis, this study proposed a new insight based on the peptide-membrane interaction for antimicrobial peptide design. The peptide electropositivity and hydrophobicity is highly related to membrane perturbation, in particular, the finding of peptide aggregates with higher electropositivity dispersing in electronegative bacterium-like membrane is a major reason to cause the higher antibacterial activity. Therefore, the tuning of peptide hydrophobicity and electropositivity for peptide aggregates in aqueous phase is an important implication for potential antibacterial peptide design.
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