Alesia McKeown

University Honors with Honors in Chemistry

Major: Chemistry

Supervisor: Paulo Almeida, Chemistry and Biochemistry

Investigation of the Effect of Salt Bridge Formation on the Vesicle Permeabilization Mechanism of Two Membrane-Active Peptides

The interaction of two mutant antimicrobial peptides with lipid vesicles were investigated in hopes of developing a better understanding of the thermodynamic effect of salt bridge formation on the mechanism of release of vesicle contents. The two mutants, Tp1 and CecAV1, were synthesized to allow for salt bridge formation and subsequent reduction of the Gibbs free energy of insertion into the bilayer. It is hypothesized that formation of salt bridges makes Tp1 and CecAV1 more efficient at translocating across the membrane relative to the original peptides, transportan 10 and cecropin A; a graded release mechanism is expected for both. Three types of fluorescence experiments were performed for each peptide to better understand the mechanism of interaction with the lipid vesicle. Efflux experiments were performed to measure the amount of dye released from encapsulated vesicles induced by binding of the peptide. Direct measurement of the on and off rates of binding of the peptide to the vesicle were investigated via fluorescence resonance energy transfer. The mechanism of release, whether graded or all-or-none, was discovered by performing the ANTS/DPX assay. It was concluded that Tp1 does not form any salt bridges and inserts into the bilayer as a monomer. It is less efficient at translocating across the membrane relative to Tp10 and causes a slightly graded release. In the case of CecAV1, one intra-molecular salt bridge is formed but the reduction in the Gibbs free energy of insertion is not significant enough to decrease it to a value below the critical threshold; an all-or-none mechanism is observed.