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 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 Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe.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.
Lauria
Graziantonio
G
Department of Pharmaco-Biology, University of Bari, Italy.
Sanchez
Paloma
P
Della Rocca
Blasco Morozzo
BM
Pierri
Ciro Leonardo
CL
Polizio
Francesca
F
Stipani
Italo
I
Desideri
Alessandro
A
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D007656:Ketoglutaric Acids / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013113:Spin Labels
D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; D013113:Spin Labels; C021723:oxoglutarate translocator