Conversion of an Alpha-Helical to a Beta-Sheet Membrane Protein Fold through Pro-Gly Mutations in the Loop of a Human CFTR Transmembrane Hairpin

Abstract

Membrane proteins adopt two fundamental types of fold in nature. Eukaryotic membranes harbor only α-helical bundles composed of series of interacting hydrophobic α-helices separated by aqueous-located loops of varying length, while β-barrel structures are found in the outer membrane of gram-negative bacteria, mitochondria and chloroplasts. Their interwoven β-sheet structures are comprised of pairs of membrane-spanning β-strand hairpins with a characteristic alternating hydrophobic/small residue sequence pattern, connected by a short intervening loop. We report that mutations in the loop of a eukaryotic α-helical hairpin peptide from the cystic fibrosis transmembrane conductance regulator (CFTR) can induce the peptide to adopt a β-sheet structure in a membrane-mimetic environment. We characterized in sodium dodecylsulfate (SDS), sodium perfluorooctanoate (SPFO) and dodecyl maltoside (DDM) micelles several constructs of the CFTR transmembrane 3 and 4 (TM3-loop-TM4) ‘hairpins’ in which Pro-Gly residues have been either inserted or substituted to promote a β-turn within the putative loop region. Suitable positioning of the Pro-Gly couplet caused the formation of a ladder of discrete oligomers on SDS-PAGE and SPFO-PAGE. Circular dichroism spectroscopy showed that mutants displaying oligomeric patterns adopt a stable β-sheet structure in detergents. In addition, size-exclusion chromatography and pyrene excimer fluorescence analysis evidenced that β-sheet oligomers are not proliferative and/or random aggregates. Such a facile change from an α-helical to an oligomeric β-hairpin structure may have implications into the evolutionary connection between α-helical and β-barrel membrane proteins. Indeed, considering that the first membrane proteins were helical hairpins (1), these results could reproduce an early differentiation event in the evolution of membrane proteins.1. Frishman, D. (2010) Structural bioinformatics of membrane proteins, 1st ed., pp xi, 281 p. Springer, New York.