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

Abstract

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 Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe.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

The mitochondrial oxoglutarate carrier: from identification to mechanism

“…In the case of OGC, several biophysical methods have been applied to characterize its structure and dynamics. Single transmembrane segments of OGC have been used in site-directed, spin-labeling experiments (Lauria et al 2008;Morozzo della Rocca et al 2003), circular dichroism and nuclear magnetic resonance in high concentrations of trifluoroethanol and sodium dodecyl sulfate (Castiglione-Morelli et al 2004. Taken together the results of these experiments are in agreement with the accepted view that OGC is likely to have a structure similar to the ADP/ATP carrier.…”

The Interaction of Hemin, a Porphyrin Derivative, with the Purified Rat Brain 2-Oxoglutarate Carrier

“…WHAT IF web server [57] was employed for assessing the goodness of both structural and energetic properties of the generated 3D comparative model. More details are available from the reference papers [58,59]. The OGC 3D structure was subsequently treated with the Protein Preparation Tool [60] of the Schrodinger suite for further checks on protonation states at physiological pH and tautomers.…”

Sequence Features of Mitochondrial Transporter Protein Families

“…Indeed, such features were later found in several other members of the protein family [31][32][33][34], along with conserved proline, glycine and acidic amino acid residues [35]. The conserved residues were later compiled into a consensus characteristic or "signature motif" for mitochondrial carriers, Px(D/E)xx(K/R) [36], and the conserved charged residues were indeed found to take part in specific conserved salt-bridge contacts, termed the matrix network [37][38][39][40][41][42], in the 3D structure of the proteins [43,44]. The signature motif can be extended in the C-terminal direction to include a proximal glutamine (Q) residue, which helps stabilize the salt-bridge contacts on the matrix side of the carrier, forming the "Q brace" [44,45].…”