Computer Simulations and Experimental Designs to Study the Mechanisms of Actions of Antimicrobial Peptides

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Computer Simulations and Experimental Designs to Study the Mechanisms of Actions of Antimicrobial Peptides

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Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics, which have a serious resistance crisis. We studied the mechanisms of three different types of AMPs using computational and experimental methods. First, computer simulations are performed to study the AMP microcin J25 (MJ25), a 21-mer peptide with an unusual lasso structure and high activity against Gram-negative bacteria. MJ25 has intracellular targets. The initial step for MJ25 acquisition in bacterial cells is binding to the outer membrane receptor FhuA. Molecular dynamics simulation is implemented to study the binding mechanism of MJ25 to FhuA and to search for important binding residues. The absolute binding free energy calculated from combined free energy perturbation (FEP) and thermodynamic integration (TI) methods agrees well with experimental data. In addition, computational mutation analysis reveals that the His5 is the key residue responsible for MJ25 and FhuA association. We find that the number of hydrogen bonds is essential for MJ25 binding to FhuA. This atomistic, quantitative insight sheds light on the mechanism of action of MJ25, and may pave a path for designing active MJ25 analogues. Second, protegrin-1 (PG-1) is a cationic arginine-rich AMP. It is widely accepted that PG-1 induces membrane disruption by forming pores that lead to cell death. However, the insertion mechanism for these highly cationic peptides into the hydrophobic membrane environment is still poorly understood at the molecular scale. It has previously been determined that the association of arginine guanidinium and lipid phosphate groups results in strong bidentate bonds that stabilize peptide-lipid complexes. It has also been suggested that arginine residues are able to drag phosphate groups as they insert inside the membrane to form a toroidal pore. However, whether bidentate bonds play a significant role in inducing a pore formation remains unclear. To investigate the role of bidentate complexes in PG-1 translocation, we conducted molecular dynamics simulations. Two computational electroporation methods were implemented to examine the translocation process. We found PG-1 could insert into the membrane provided the external electric potential is large enough to first induce a water column or pore within the lipid bilayer membrane. We also found that the highly charged PG-1 is capable in itself of inducing molecular electroporation. Substitution of arginines with charge-equivalent lysines showed a markedly reduced tendency for insertion. This indicates the guanidinium group likely facilitates PG-1 translocation. Potential of mean force calculations suggest that peptide insertion inside the hydrophobic environment of the membrane core is not favored. We found that formation of a water column or pore might be a prerequisite for PG-1 translocation. We also found PG-1 can stabilize the pore after insertion. We suggest that PG-1 could be a pore inducer and stabilizer. This work sheds some light on PG-1 translocation mechanisms at the molecular level. Methods presented in this study may be extended to other arginine-rich antimicrobial and cell-penetrating peptides. Third, oncocin is a proline-rich antimicrobial peptide that inhibits protein synthesis by binding to the bacterial ribosome. In this work, the antimicrobial activity of oncocin was improved by systematic peptide mutagenesis and activity evaluation. We found that a pair of cationic substitutions (P4K and L7K/R) improves the activity by 2-4 fold (p<0.05) against multiple Gram-negative bacteria. An in vitro transcription / translation assay indicated that the increased activity was not because of stronger ribosome binding. Rather a cellular internalization assay revealed a higher internalization rate for the optimized analogs thereby suggesting a mechanism to increase potency. In addition, we found that the optimized peptides’ benefit is dependent upon nutrient depleted media conditions. The molecular design and characterization strategies have broad potential for development of antimicrobial peptides.


University of Minnesota Ph.D. dissertation. October 2018. Major: Chemical Engineering. Advisor: Benjamin Hackel. 1 computer file (PDF); xi, 128 pages.

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Lai, Pin-Kuang. (2018). Computer Simulations and Experimental Designs to Study the Mechanisms of Actions of Antimicrobial Peptides. Retrieved from the University Digital Conservancy,

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