Mechanisms of Pore Formation in Membranes
Description
Non-receptor-mediated pore-forming peptides and proteins can be found in most living organisms. Differences in their pore-forming mechanisms can be explained by differences in their peptide-lipid and peptide-peptide interactions. Most antimicrobial peptides (AMPs) form transient pores in membranes by a mechanism that is called the 'carpet model.' This antimicrobial host defense function is achieved by cationic/amphipathic peptides which bind and insert into the negatively-charged phospholipid head groups of microbial membrane, thereby disrupting the lipid bilayer. Toxins and venoms, on the other hand, form long-lived pores in lipid bilayers in a mechanism called the 'barrel-stave' model. In this model, peptides bind to the membrane and oligomerize to form long-lived, transmembrane pores. The available tools to characterize the pore-forming mechanisms of AMPs and toxins are limited. In this work, a set of novel fluorescence-based assays were developed to differentiate between the aforementioned mechanisms. The assays were tested with a set of known AMPs and pore-forming toxins. Further experiments were developed to help understand the unexpected dynamic activity of the fungal toxin alamethicin. Finally, the techniques developed here were used in two high-throughput screens. In the first screen, an iterative library of beta-sheet rich peptides, based on a previously discovered AMP, was screened for highly potent long lived pores. Second, a library based on the pore-forming toxin melittin, the main component of European honey bee venom, was screened for gain-of-function and loss of function analogs. The screen found a high frequency of conserved residues required for melittin's activity and also some substitutions that significantly enhanced pore-formation. A single substitution was also discovered that completely knocked-out melittin's hemolytic and pore-forming activity