As antibiotics have been developed and widely used over time, there has been increasing levels of antibiotic resistance within bacterial pathogens. Antimicrobial peptides (AMPs) are promising alternatives to antibiotics because they effectively kill bacteria with a lower threat of resistance, due to their targeting general cell structures instead of particular molecular targets. Our project focuses on buforin II (BF2), an amphipathic, positively-charged AMP that translocates across the bacterial cell membrane and binds nonspecifically to DNA as a likely part of its mode of action. We hypothesize that increasing the peptide’s net positive charge via site-specific rational arginine substitution will increase binding affinity to its membrane and DNA targets, both of which are negatively charged. Once this has been achieved, we can then investigate how binding affinity relates to overall AMP potency. While other ongoing work is focused on the effect of arginine substitutions on BF2/DNA interactions, the work presented here focuses on BF2’s interactions with the cellular membrane, evaluating how arginine mutations affect its membrane binding affinity, and ultimately observing subsequent effects on antimicrobial potency. This is achieved through the following workflow: (1) initial, replicate molecular dynamics (MD) simulations of the wildtype BF2/membrane system to generate a conformational ensemble, (2) partial, constrained charge optimization, a technique used to find a hypothetical molecular charge distribution for optimal binding affinity, to efficiently screen potential residues for arginine substitution, (3) replicate MD simulations of explicit candidate mutants suggested by step (2) to generate mutant conformational ensembles, (4) continuum electrostatic calculations to evaluate the electrostatic binding free energy of these mutants, and (5) experimental testing of mutants for target binding affinity and potency. Having focused thus far on the initial steps of the workflow, here we report preliminary data toward these designs. Our work will ultimately provide insight into the relationship between AMP/membrane interactions and potency to enable more effective therapeutics in the future.

Targeted buforin II arginine substitution: Progress towards better cellular membrane binding

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