Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling

Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising thesixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteineshave been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine andsubsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have beenassayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidthanalysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 toI298, indicating that it has an a-helical structure. Saturation behaviour, in presence of paramagnetic perturbants of differenthydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect tothe lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activitydata indicate that the segment is made by an alpha helix, accommodated in an amphipathic environment, partially distortedin the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region ofthe segment is less restrained and more flexible than the N-terminus.