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

Fourier transform infrared spectroscopy has been applied to the study of th e carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, either solubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes. Its secondary structure ha s been estimated by means of Fourier self-deconvolution followed by curve f it. A Voigt function was used to fit the components of the deconvoluted spe ctrum, aiming to account for any distortions introduced by deconvolution. F or 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 pl us unordered structures, coherent with the six transmembrane spanning helix model. Moreover, the problem of structure preservation on drying was addre ssed, and several observations pointed to a maintenance of the protein stru cture in dry films. Comparison of reconstituted and solubilized samples ind icated the presence of both lipid-induced changes in the protein (decrease of the beta -sheets and increase of unordered structures) and protein-induc ed changes in the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of alpha -helix and unordered structure cont ributions for the reconstituted form, H/D exchange experiments were perform ed. Between 35% and 45% of the amino acids were finally assigned to alpha - helix structures, compatible with the existence of five or six transmembran e spanning helices in the transporter. The level of H/D exchange was determ ined after 15 h of exposure to D2O vapor to be 85%, reflecting a high acces sibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.