Structure and Function of Direct Delivery Peptides - Summary Classical concepts of protein folding in membranes were elucidated many years ago and can now be found in textbooks. Yet, we have observed self-assembled peptide nanopore structures, with many potential applications, that cannot be fully explained by these classical concepts. The self-assembled helical nanopores, form macromolecule-sized pores in bilayers with diameters greater than 5 nm. The peptides appear not to be hydrophobic enough to insert into membrane-spanning structures, yet they do exactly that. We will test the idea that nanopore stability in the bilayer can be explained by cooperative H-bond networks between peptides, lipids, and water on the interior of the pore, in addition to classical hydrophobic interactions with the bilayer. The nanopores we will study are formed through the self-assembly of helical peptides that have been evolved through synthetic molecular evolution (generations of iterative library design and screening). Elucidating the nonclassical interactions that govern the self-assembly and stabilization of these nanopores will reveal fundamentally novel principles of the structure and self-assembly of amphipathic peptides and proteins in membranes and will be the first quantitative characterization of the role of expansive H-bond networks in the stabilization of transmembrane pores. In the proposed work, we will quantify i) how peptide nanopore structure and function are affected by targeted substitution of critical residues in the H-bond network and putative pH-sensor, and ii) how peptide nanopore structure and function are affected by changes in peptide hydrophobic residues and membrane physical properties, as defined by lipid composition. Atomistic molecular dynamics simulations will be used throughout this work to guide the design and the interpretation of experiments. By understanding the sequence-structure- function relationships of nanopore formation, including pH-sensing and membrane selectivity, we will enable future nanopore optimization for specific applications. We envision usage in biotechnology and medicine, for facile protein therapy, drug or macromolecule delivery, anticancer activity, and more.
