title: A CD study of uncoupling protein-1 and its transmembrane and matrix-loop domains.
abstract: Conformations of the prototypic UCP-1 (uncoupling protein-1) and its TM (transmembrane) and ML (matrix-loop) domains were studied by CD spectroscopy. Recombinant, untagged mouse UCP-1 and a hexahistidine-tagged version of the protein were obtained in high purity following their overexpression in Escherichia coli. The TM and ML domains of hamster UCP-1 were chemically synthesized. Conformations of both recombinant UCP-1 proteins were dominantly helical (40-50%) in digitonin micelles. Binding of the purine nucleotides GDP and GTP to UCP-1, detected in the near-UV CD region, supported the existence of the functional form of the protein in digitonin micelles. All individual TM and ML peptides, except the third ML domain, adopted helical structures in aqueous trifluoroethanol, which implies that, in addition to six TM segments, at least two of the ML domains of the UCP-1 can form helical structures in membrane interface regions. TM and ML domains interacted with vesicles composed of the main phospholipids of the inner membrane of mitochondria, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, to adopt dominantly beta- and/or unordered conformations. Mixtures of UCP-1 peptide domains spontaneously associated in aqueous, phospholipid vesicles and digitonin micelle environments to form ordered conformations, which exhibited common features with the conformations of the full-length proteins. Thermal denaturations of UCP-1 and its nine-peptide-domain assembly in digitonin were co-operative but not reversible. Assembly of six TM domains in lipid bilayers formed ion-conducting units with possible helical bundle conformations. Consequently, covalent connection between peptide domains, tight domain interactions and TM potential are essential for the formation of the functional conformation of UCP-1.
authors.affiliation: Department of Chemistry, Wilfrid Laurier University, Waterloo, Ontario, Canada N2L 3C5. mjelokhani@wlu.ca
authors.forename: Masoud
authors.initials: M
authors.lastname: Jelokhani-Niaraki
authors.forename: Marina V
authors.initials: MV
authors.lastname: Ivanova
authors.forename: Bonnie L
authors.initials: BL
authors.lastname: McIntyre
authors.forename: Cheryl L
authors.initials: CL
authors.lastname: Newman
authors.forename: Fern R
authors.initials: FR
authors.lastname: McSorley
authors.forename: Elizabeth K
authors.initials: EK
authors.lastname: Young
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006224:Cricetinae; D004594:Electrophysiology; D015870:Gene Expression; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008647:Mesocricetus; D051379:Mice; D008823:Micelles; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature; D000071256:Uncoupling Protein 1
chemical_list: D007473:Ion Channels; D008823:Micelles; D024101:Mitochondrial Proteins; D010446:Peptide Fragments; C000604372:Ucp1 protein, mouse; D000071256:Uncoupling Protein 1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate