Ion Channel Design Using Unnatural Amino Acids

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Nearly a third of completed genomic sequences and almost half of all receptors that are likely to be targets for drug design are integral membrane proteins. Understanding the structure and energetics of membrane proteins is therefore a key and unsolved problem in structural biology. In contrast to proteins soluble in aqueous media, the primary interactions that contribute to the stability and specificity of membrane protein structures are poorly understood. Soluble proteins display a bipartite architecture with hydrophilic exteriors and hydrophobic interiors. This kind of binary patterning is not observed in membrane proteins, rendering design a difficult problem. The challenge is one of control over structure in nonpolar surroundings. The broad long term objective of this proposal is the development of design elements for membrane proteins. Our approach relies on the unique phase separation properties of highly fluorinated materials. The proposed studies will make use of folding driven by phase separation in the non polar membrane environment of appropriately positioned fluorinated side chains to deliver predetermined structural and functional ensembles. The specific aims of this proposal are: (1) To develop methodology for synthesis of enantiomerically pure fluorinated amino acid analogues; (2) To probe the three dimensional structure of fluorous peptides using biophysical techniques; (3) To study the effect of stability of fluorous phases in protein environments by selectively replacing core hydrophobic residues; (4) design and synthesis of membrane spanning and pore forming peptide ensembles based on phase separation within the membrane; (5) biophysical characterization of the peptide-lipid interactions in lipid vesicles and planar lipid membranes using a combination of fluorescence and CD spectroscopy, differential scanning and isothermal titration calorimetry; (6) characterization of the influence of peptides on lipid morphology and direct visualization of pore formation by scanning force microscopy and (7) investigation of channel activity using single channel conductance and fluorescence assays probing dye or H+ release. Ultimately the design and characterization studies proposed here should facilitate the construction of effective membrane transport agents that will add to currently available antibiotics and help in combating the ever- increasing resistance of bacteria to current therapeutic drugs.