The oxoglutarate/malate carrier (OGC), a transport protein of the inner mitochondrial membranes, catalyses the exchange of 2-oxoglutarate for malate and plays a central role in several metabolic processes [Runswich,M.J.,Walker,J.E.,Bisaccia,F., Iacobazzi,V.,and Palmieri,F.(1990) Biochemistry 29, 11033–11040]. In order to understand the mechanism of oxoglutarate translocation through the OGC protein, the elucidation of the carrier structure is essential. The crystallization of transport proteins has been difficult because they are hydrophobic, show a strong tendency to aggregate and undergo the rapid structural changes required for their catalytic cycle. Among the members of the mitochondrial carriers family, only the X-ray crystallographic structure of the carboxyatractyloside-ADP/ATP carrier complex has been determined [Pebay-Peyroula,C.E.,- Dahou-Gonzales,C.,Kahn,R.,Trezeguet,V.,Lauquin,G.J.M.,and Brandolin, G.(2003)Nature 426, 39–43]. We studied the structure of the first two transmembrane segments of the bovine mitochondrial OGC by CD and NMR spectroscopies. The synthesis of the peptides was performed on a solid-phase automatic peptide synthesiser, Pioneer TMPeptide Synthesis System. The Fmoc strategy was used. The peptides were purified by HPLC and the purity was assessed by mass spectroscopy. Circular dichroism spectra were recorded with a JASCO 600A automatic circular dichrograph. NMR measurements were carried out on a VARIAN Inova 500 NMR spectrometer. Two-dimensional TOCSY and NOESY spectra were acquired in the phase-sensitive mode.CD data showed that at high concentrations of TFE(>50%) and SDS (>2%) peptides 21–46 and 78–108 assume a-helical structure. NMR data showed a well-defined a-helix in the region K24-V39 of peptide 21–46 and in the region A86-F106 of peptide 78–108. The helix of peptide 21–46 is essentially hydrophobic, whereas that of peptide 78–108 is predominantly hydrophilic.Our helical structures were superimposed on the structure of helices H1 and H2 of the ADP/ATP carrier (PDB accession code: 1okc). The good agreement between the solution structure of our peptides and the crystal structure of the first two transmembrane segments of the ADP/ATP carrier suggests that, in spite of the low sequence homology between them, the structure of OGC may be similar to that of the ADP/ATP carrier. Studies are in progress on the other transmembrane segments and loop regions to test if this conclusion may apply to other regions of the OGC.
CD and NMR studies of transmembrane segments of the mitochondrial oxoglutarate carrier
LAURIA, GRAZIANTONIO;
2004-01-01
Abstract
The oxoglutarate/malate carrier (OGC), a transport protein of the inner mitochondrial membranes, catalyses the exchange of 2-oxoglutarate for malate and plays a central role in several metabolic processes [Runswich,M.J.,Walker,J.E.,Bisaccia,F., Iacobazzi,V.,and Palmieri,F.(1990) Biochemistry 29, 11033–11040]. In order to understand the mechanism of oxoglutarate translocation through the OGC protein, the elucidation of the carrier structure is essential. The crystallization of transport proteins has been difficult because they are hydrophobic, show a strong tendency to aggregate and undergo the rapid structural changes required for their catalytic cycle. Among the members of the mitochondrial carriers family, only the X-ray crystallographic structure of the carboxyatractyloside-ADP/ATP carrier complex has been determined [Pebay-Peyroula,C.E.,- Dahou-Gonzales,C.,Kahn,R.,Trezeguet,V.,Lauquin,G.J.M.,and Brandolin, G.(2003)Nature 426, 39–43]. We studied the structure of the first two transmembrane segments of the bovine mitochondrial OGC by CD and NMR spectroscopies. The synthesis of the peptides was performed on a solid-phase automatic peptide synthesiser, Pioneer TMPeptide Synthesis System. The Fmoc strategy was used. The peptides were purified by HPLC and the purity was assessed by mass spectroscopy. Circular dichroism spectra were recorded with a JASCO 600A automatic circular dichrograph. NMR measurements were carried out on a VARIAN Inova 500 NMR spectrometer. Two-dimensional TOCSY and NOESY spectra were acquired in the phase-sensitive mode.CD data showed that at high concentrations of TFE(>50%) and SDS (>2%) peptides 21–46 and 78–108 assume a-helical structure. NMR data showed a well-defined a-helix in the region K24-V39 of peptide 21–46 and in the region A86-F106 of peptide 78–108. The helix of peptide 21–46 is essentially hydrophobic, whereas that of peptide 78–108 is predominantly hydrophilic.Our helical structures were superimposed on the structure of helices H1 and H2 of the ADP/ATP carrier (PDB accession code: 1okc). The good agreement between the solution structure of our peptides and the crystal structure of the first two transmembrane segments of the ADP/ATP carrier suggests that, in spite of the low sequence homology between them, the structure of OGC may be similar to that of the ADP/ATP carrier. Studies are in progress on the other transmembrane segments and loop regions to test if this conclusion may apply to other regions of the OGC.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
