The secondary structure of the inhibited mitochondrial ADP/ATP transporter from yeast analyzed by FTIR spectroscopy.
Fourier transform infrared spectroscopy has been applied to the study of the carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, either solubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes. Its secondary structure has been estimated by means of Fourier self-deconvolution followed by curve fit. A Voigt function was used to fit the components of the deconvoluted spectrum, aiming to account for any distortions introduced by deconvolution. For any of the states analyzed, reconstituted or solubilized, in solution or in dry films, 60-70% of the amino acids are found to adopt alpha-helix plus unordered structures, coherent with the six transmembrane spanning helix model. Moreover, the problem of structure preservation on drying was addressed, and several observations pointed to a maintenance of the protein structure in dry films. Comparison of reconstituted and solubilized samples indicated the presence of both lipid-induced changes in the protein (decrease of the beta-sheets and increase of unordered structures) and protein-induced changes in the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of alpha-helix and unordered structure contributions for the reconstituted form, H/D exchange experiments were performed. Between 35% and 45% of the amino acids were finally assigned to alpha-helix structures, compatible with the existence of five or six transmembrane spanning helices in the transporter. The level of H/D exchange was determined after 15 h of exposure to D(2)O vapor to be 85%, reflecting a high accessibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.
Lórenz
V A
VA
Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Villaverde
J
J
Trézéguet
V
V
Lauquin
G J
GJ
Brandolin
G
G
Padrós
E
E
D000577:Amides / Q000378:metabolism; D001278:Atractyloside / Q000031:analogs & derivatives / Q000737:chemistry; D003902:Detergents; D017666:Deuterium Oxide / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D005960:Glucosides / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008823:Micelles; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology*; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D016011:Normal Distribution; D010743:Phospholipids / Q000378:metabolism; D017433:Protein Structure, Secondary; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data
D000577:Amides; D003902:Detergents; D004791:Enzyme Inhibitors; D005960:Glucosides; D008051:Lipid Bilayers; D008823:Micelles; D010743:Phospholipids; D011522:Protons; D001278:Atractyloside; D006639:Histidine; C040358:dodecyl maltoside; D000226:Mitochondrial ADP, ATP Translocases; D017666:Deuterium Oxide; C003853:carboxyatractyloside