The Secondary Structure of the Inhibited Mitochondrial ADP/ATP Transporter from Yeast Analyzed by FTIR Spectroscopy
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- 作者：Ví ; ctor A. Ló ; renz ; Joaquim Villaverde ; Vé ; ronique Tré ; zé ; guet ; Guy J.-M. Lauquin ; Gé ; rard Brandolin ; and Esteve Padró ; s
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：July 31, 2001
- 年：2001
- 卷：40
- 期：30
- 页码：8821 - 8833
- 全文大小：132K
- 年卷期：v.40,no.30(July 31, 2001)
- ISSN：1520-4995
文摘
Fourier transform infrared spectroscopy has been applied to the study of the carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, eithersolubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes. Its secondary structurehas been estimated by means of Fourier self-deconvolution followed by curve fit. A Voigt function wasused to fit the components of the deconvoluted spectrum, aiming to account for any distortions introducedby 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 
-helix plus unordered structures, coherent with the sixtransmembrane spanning helix model. Moreover, the problem of structure preservation on drying wasaddressed, 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 changesin the protein (decrease of the 
-sheets and increase of unordered structures) and protein-induced changesin the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of -helixand 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 -helix structures, compatible withthe existence of five or six transmembrane spanning helices in the transporter. The level of H/D exchangewas determined after 15 h of exposure to D2O vapor to be 85%, reflecting a high accessibility of theamide hydrogens even for the carboxyatractyloside-inhibited state.
-helix plus unordered structures, coherent with the sixtransmembrane spanning helix model. Moreover, the problem of structure preservation on drying wasaddressed, 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 changesin the protein (decrease of the -sheets and increase of unordered structures) and protein-induced changesin the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of -helixand 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 -helix structures, compatible withthe existence of five or six transmembrane spanning helices in the transporter. The level of H/D exchangewas determined after 15 h of exposure to D2O vapor to be 85%, reflecting a high accessibility of theamide hydrogens even for the carboxyatractyloside-inhibited state.