The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.
To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.
Department of Biochemistry & Molecular Biology, FUHS/The Chicago Medical School, North Chicago, Illinois 60064, USA.
R S
RS
Kaplan
J A
JA
Mayor
R
R
Kotaria
D E
DE
Walters
H S
HS
McHaourab
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics*; D002614:Chelating Agents / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008928:Mitochondria / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012441:Saccharomyces cerevisiae; D012997:Solvents; D013113:Spin Labels
D002352:Carrier Proteins; D002614:Chelating Agents; D005656:Fungal Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D012997:Solvents; D013113:Spin Labels; C020723:citrate-binding transport protein; C026058:EDDA; D004492:Edetic Acid; D010100:Oxygen