There are many natural and designed peptides that permeabilize membranes, and there are multiple mechanisms by which membrane permeabilization can occur. Yet, peptides that unequivocally self-assemble into equilibrium, membrane-spanning pores at low peptide to lipid ratios (P:L�1:1000) are very rare. The design and engineering of such peptide �nanopores� in lipid bilayer membranes is desirable as it could lead to improved biosensor platforms, targeted therapeutics, exogenous ion channels,  or drug delivery vehicles. While the few well studied pore-forming peptides have provided a lot of information about the architecture of peptide pores, especially a-helical pores, our knowledge of the fundamental molecular principles of pore formation is not detailed enough for rational engineering. This is a roadblock to the design of new pore-forming peptides and to the optimization of known pore-formers for particular applications. In this work we show how novel, highly potent, equilibrium pore-forming peptides can be discovered using synthetic molecular evolution, i.e. iterative cycles of combinatorial library design and high-throughput screening. In the first example, we used two generations of de novo library design and screening to identify highly potent pore-formers that self-assemble into b-sheets in membranes. These peptides may be the only known examples of highly potent, pore-forming peptides that have b-sheet secondary structure in membranes. In the second example we designed an iterative library that used the helical pore-former melittin as a template. From this library we identified gain-of-function pore-formers that are much more potent than melittin. The results demonstrate the power of synthetic molecular evolution for the discovery and engineering of membrane active peptides.

Discovering highly potent pore-forming peptides using synthetic molecular evolution

245th National Meeting (2013)

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