The secondary structure of the inhibited mitochondrial ADP/ATP transporter from yeast analyzed by FTIR spectroscopy

V. A. Lórenz, J. Villaverde, V. Trézéguet, G. J.M. Lauquin, G. Brandolin, E. Padrós

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11 Citations (Scopus)

Abstract

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 α-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 β-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 α-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 α-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 D2O vapor to be 85%, reflecting a high accessibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.
Original languageEnglish
Pages (from-to)8821-8833
JournalBiochemistry
Volume40
DOIs
Publication statusPublished - 31 Jul 2001

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