The amide I changes accounted for 15% of the amide I area, and so 7

The amide I changes accounted for 15% of the amide I area, and so 7.5% of the protein (25 amino acids) was involved in the structural changes. tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed. INTRODUCTION The ADP/ATP transporter is located in the inner mitochondrial membrane, from where it mediates the exchange of cytosolic ADP for ATP generated in the mitochondria. The transporter adopts two structural conformations, which can be detected by its characteristic sensitivity to inhibitors. In the so-called CATR conformation the transporter can be blocked by atractyloside (atr) and carboxyatractyloside (c-atr) acting from the cytosolic side, whereas in the BA conformation the bongkrekic acid (BA) and isobongkrekic acid block the transporter from the matrix side. Both conformations show particular chemical, immunochemical, and enzymatic reactivities, and their interconversions are probably a key feature of the transport process. For further details, see reviews by Brandolin et al. (1993a), AZ-960 Fiore et al. (1998), and Kaplan (1996). Most of the knowledge about the ADP/ATP transporter has been obtained in experiments performed on mitochondria. In this way, valuable information concerning its function and indirect information about the structural changes involved in the CATR to BA conformation transition has been obtained. However, direct structural information about the ADP/ATP transporter is usually scarce to date. Spectroscopic methods can supply part of this information which is currently lacking, provided that the protein is usually obtained highly pure and in SFTPA2 a well-defined conformation. Spectroscopic studies AZ-960 of the ADP/ATP transporter have encountered one major problem: its instability during the purification process (Klingenberg et al., 1995). Since the ADP/ATP transporter is usually a membrane protein, purification is performed through a solubilized state. In studies performed in very fresh preparations of the solubilized protein, only half of its substrate binding sites are retained (Brandolin et al., 1993b; Kr?mer and Klingenberg, 1977). Therefore, the solubilized and unliganded ADP/ATP transporter contains a large number of inactive molecules which increase with the time the sample spends solubilized, until reaching full inactivation in a matter of a few hours. The carrier which has lost its capacity to bind ligands in AZ-960 a time-dependent manner AZ-960 will be referred to as (Kr?mer and Klingenberg, 1977). Once reconstituted into liposomes, the transporter remains stable for many hours (Brandolin et al., 1980; Klingenberg et al., 1995). To reduce the time the transporter spends solubilized, the purification procedure can be simplified, so that the reconstituted transporter is usually obtained only partially purified (50% of contaminating proteins; see Heidk?mper et al., 1996; Klingenberg et al., 1995). Obviously, this preparation would not be AZ-960 suitable for spectroscopic analysis. The high instability of the solubilized ADP/ATP transporter entails some questions. Why is it so unstable in the solubilized state? Is the instability related to its function? Which structural changes are responsible for the reduction in the number of binding sites during its isolation? In this work, Fourier transform infrared (FTIR) spectroscopy is used, aiming at characterizing the structural changes responsible for the reduction of binding sites during purification of the yeast ADP/ATP transporter from (Anc2pHis; Fiore et al., 2000). FTIR spectra of proteins contain structural information, mainly encoded in band positions of the amide I, but also in the amide II and amide A vibrations (Bandekar, 1992; Goormaghtigh et al., 1994a; Krimm and Bandekar, 1986). Several guides to assign secondary structure from the position of the amide I components have been published; see Arrondo et al. (1993), Goormaghtigh et al. (1994b), and Tamm and Tatulian (1997). Theoretically, by comparing FTIR spectra of time-inactivated Anc2pHis and fully functional, noninhibited Anc2pHis, we could have some insights into the structural.