5and6). greater accessibility to the solvent on the matrix side, whereas the CATR-bAnc1p complex is more accessible on the intermembrane side. These results are discussed with respect to the structural and biochemical data available on Ancp. Keywords:Mass Spectrometry (MS), Membrane Enzymes, Membrane Proteins, Metabolism, Mitochondria, Mitochondrial Transport, ADP/ATP Carrier, Conformational Dynamics, H/D Exchange, Mitochondrial Carrier == Introduction == Import and export of metabolites across mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner mitochondrial membrane. Proteins of the mitochondrial carrier family (MCF)5are embedded in this membrane, and each member of the family achieves the selective transport of specific metabolites (1). Among these, the ADP/ATP carrier (Ancp) transports ADP into the mitochondrial matrix and exports newly synthesized ATP toward the cytosol. Mainly due to its natural abundance, the ADP/ATP carrier is the best characterized within the MCF (2). The mitochondrial ADP/ATP exchange process can be blocked by two specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). CATR and BA bind with high affinity to two distinct pre-existing conformations of the carrier referred to as CATR and BA conformations, respectively, resulting in the formation of stable CATR- and BA-carrier complexes. It was suggested that the transition between the CATR and BA conformations is similar to that involved in the ADP/ATP transport (2). Members of MCF share common features,i.e.similar molecular masses of about 30 kDa, a so-called tripartite organization consisting of three sequence repeats of about 100 amino acid residues each, and the presence of the conserved motif PX(D/E)XX(K/R) in each repeat (1). Additionally, the Ancp have a common signature sequence, RRRMMM, that is absent in other MCF members (3). The intrinsic mechanisms of ADP/ATP transport, nucleotide recognition, and Ancp conformational changes have been widely investigated mainly with the bovine isoform 1 (bAnc1p) and theSaccharomyces cerevisiaeisoform 2 (ScAnc2p). The large body of biochemical and biophysical data available demonstrated that CATR- and BA-carrier complexes display distinct structural features (4). Understanding the mechanism of transport proteins in biological membranes at the molecular level requires high resolution structural information that is usually obtained from x-ray crystallography and/or TNF-alpha NMR spectroscopy studies. The three-dimensional structure of bAnc1p locked with CATR has been solved at 2.2 resolution by x-ray crystallography (5). In this structure the six transmembrane helices of bAnc1p called H1 to H6 form a cavity JNJ-17203212 with a deep, cone-shaped depression accessible only from the cytosolic side (seeFig. 1). Binding of CATR in the cavity blocks Ancp, and numerous studies have suggested that CATR- and ADP-binding sites overlap at least JNJ-17203212 partially (4). In each odd-numbered helix, JNJ-17203212 the proline of the MCF motif introduces a sharp kink, which is suggested to act as a hinge in the straightening out the helices when bAnc1p is open to the matrix side (5). The connections between the even- and JNJ-17203212 odd-numbered helices are made by the intermembrane space (IMS) loops C1 and C2 and by matrix loops M1, M2, and M3 including short -helical stretches h12, h34, and h56, respectively (seeFig. 1). The latter are parallel to the membrane surface and strengthen the closed conformation of the CATR-carrier complex on the matrix side (Fig. 1). In contrast to the CATR-bound bAnc1p, the structure of the bovine BA inhibited form still remains unknown. Its characterization, however, would significantly extend our understanding of JNJ-17203212 the ADP/ATP transport.