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Conversion of a Porin-like Peptide Channel into a Gramicidin-like Channel by Glycine to D-alanine Substitutions

Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202

Correspondence: Address reprint requests to Lili Guo, Dept. of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202. Tel.: 317-274-7507; Fax: 317-274-4686; E-mail: lguo{at}iupui.edu.

The ß-barrel and ß-helix formation, as in porins and gramicidin, respectively, represent two distinct mechanisms for ion channel formation by ß-sheet proteins in membranes. The design of ß-barrel proteins is difficult due to incomplete understanding of the basic principles of folding. The design of gramicidin-like ß-helix relies on an alternating pattern of L- and D-amino acid sequences. Recently we noticed that a short ß-sheet peptide (xSxG)₆, can form porin-like channels via self-association in membranes. Here, we proposed that glycine to D-alanine substitutions of the N-formyl-(xSxG)₆ would transform the porin-like channel into a gramicidin-like ß¹²-helical channel. The requirement of an N-formyl group for channel activity, impermeability to cations with a diameter >4 Å, high monovalent cation selectivity, and the absence of either voltage gating or subconductance states upon D-alanine substitution support the idea of a gramicidin-like channel. Moreover, the circular dichroism spectrum in membranes is different, indicating a change in regular ß-sheet backbone structure. The conversion of a complex porin-like channel into a gramicidin-like channel provides a link between two different mechanisms of ß-sheet channel formation in membranes and emphasizes the importance of glycine and D-amino acid residues in protein folding and function and in the engineering of ion channels.