title: Interhelical packing in detergent micelles. Folding of a cystic fibrosis transmembrane conductance regulator construct.
abstract: Using a helix-loop-helix construct consisting of the adjacent transmembrane segments 3 and 4 of the cystic fibrosis transmembrane conductance regulator (CFTR) labeled with pyrene at both N and C termini, we describe a system for the study of intramolecular helix-helix interactions within a polytopic membrane protein. Through measurement of pyrene excimer band intensity as a determinant of helix-helix proximity, we show that the helices retain tertiary contacts in detergent micelles. Notably, the nature of the micellar detergent can alter the stability of these contacts, with perfluorooctanoate highly supportive, lysophosphatidylcholine and lysophosphatidylglycerol somewhat less tolerant, and SDS largely intolerant of such interactions. This construct is further employed to study the role of the acyl chain length of micellar detergents in modulating interhelical packing; detergents having acyl chains of 9 carbons display the greatest extent of helical packing. These results provide important information regarding the role of lipids on membrane protein folding and conformation as well as demonstrate the usefulness of a pyrene-based system in studying the forces that govern interhelical packing.
authors.affiliation: Division of Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto M5G 1X8, Canada.
authors.forename: Alex G
authors.initials: AG
authors.lastname: Therien
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000502:physiology*; D003902:Detergents; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008823:Micelles; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence
chemical_list: C505032:CFTR protein, human; D003902:Detergents; D008823:Micelles; D010446:Peptide Fragments; D019005:Cystic Fibrosis Transmembrane Conductance Regulator