title: Infrared spectroscopic studies of detergent-solubilized uncoupling protein from brown-adipose-tissue mitochondria.
abstract: The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.
authors.affiliation: Department of Biochemistry, Faculty of Science, University of the Basque Country, Bilbao, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Rial
authors.forename: A
authors.initials: A
authors.lastname: Muga
authors.forename: J M
authors.initials: JM
authors.lastname: Valpuesta
authors.forename: J L
authors.initials: JL
authors.lastname: Arrondo
authors.forename: F M
authors.initials: FM
authors.lastname: Goñi
mesh_terms: D002001:Adipose Tissue, Brown / Q000032:analysis*; D000577:Amides / Q000302:isolation & purification; D000818:Animals; D002352:Carrier Proteins; D006224:Cricetinae; D007473:Ion Channels; D008565:Membrane Proteins / Q000302:isolation & purification*; D008647:Mesocricetus; D008823:Micelles; D008928:Mitochondria / Q000032:analysis*; D000226:Mitochondrial ADP, ATP Translocases / Q000302:isolation & purification; D024101:Mitochondrial Proteins; D017830:Octoxynol; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011489:Protein Denaturation; D012689:Sequence Homology, Nucleic Acid; D013055:Spectrophotometry, Infrared / Q000379:methods; D013696:Temperature; D000071256:Uncoupling Protein 1
chemical_list: D000577:Amides; D002352:Carrier Proteins; D007473:Ion Channels; D008565:Membrane Proteins; D008823:Micelles; D024101:Mitochondrial Proteins; D000071256:Uncoupling Protein 1; D011092:Polyethylene Glycols; D017830:Octoxynol; D000226:Mitochondrial ADP, ATP Translocases