Lear, James D
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PublicationComputational Design of Water-Soluble Analogues of the Potassium Channel KcsA(2004-02-17) Lear, James D; Slovic, Avram Michael; Saven, Jeffery G.; Kono, Hidetoshi; DeGrado, William FAlthough the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield inEscherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker. PublicationOligomerization of Fusogenic Peptides Promotes Membrane Fusion by Enhancing Membrane Destabilization(2004-01-01) Lear, James D; Lau, Wai Leung; Hammer, Daniel A; Ege, David S; DeGrado, William FA key element of membrane fusion reactions in biology is the involvement of specific fusion proteins. In many viruses, the proteins that mediate membrane fusion usually exist as homotrimers. Furthermore, they contain extended triplehelical coiled-coil domains and fusogenic peptides. It has been suggested that the coiled-coil domains present the fusogenic peptide in a conformation or geometry favorable for membrane fusion. To test the hypothesis that trimerization of fusogenic peptide is related to optimal fusion, we have designed and synthesized a triple-stranded coiled-coil X31 peptide, also known as the ccX31, which mimics the influenza virus hemagglutinin fusion peptide in the fusion-active state. We compared the membrane interactive properties of ccX31 versus the monomeric X31 fusogenic peptide. Our data show that trimerization enhances peptide-induced leakage of liposomal contents and lipid mixing. Furthermore, studies using micropipette aspiration of single vesicles reveal that ccX31 decreases lysis tension, τlysis, but not area expansion modulus, Ka, of phospholipid bilayers, whereas monomeric X31 peptide lowers both τlysis and Ka. Our results are consistent with the hypothesis that oligomerization of fusogenic peptide promotes membrane fusion, possibly by enhancing localized destabilization of lipid bilayers.