The structural and dynamic features of the fourth transmembrane segment of the mitochondrial oxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance (EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutive residues from S184 to S201 which are believed to form the transmembrane segment IV were substituted individually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutants exhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined by EPR as a function of the incident microwave power in the presence and absence of two paramagnetic perturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. The periodicity of the sequence-specific variation in the spin-label mobility and the O2 accessibility parameters unambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier as an α-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicating that the helix is amphipatic, with the hydrophilic face containing the residues found to be important for transport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed, as indicated by the values of normalized mobility, which also suggest that the conformational changes occurring during transport probably involve the N-terminal region of the helix.

MOROZZO DELLA ROCCA, B., Lauria, G., Venerini, F., Palmieri, L., Polizio, F., Capobianco, L., et al. (2003). The mitochondrial oxoglutarate carrier: Structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling. BIOCHEMISTRY, 42, 5493-5499 [10.1021/bi027025q].

The mitochondrial oxoglutarate carrier: Structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling

MOROZZO DELLA ROCCA, BLASCO;POLIZIO, FRANCESCA;PEDERSEN, JENS ZACHO;DESIDERI, ALESSANDRO;
2003-01-01

Abstract

The structural and dynamic features of the fourth transmembrane segment of the mitochondrial oxoglutarate carrier were investigated using site-directed spin labeling and electron paramagnetic resonance (EPR). Using a functional carrier protein with native cysteines replaced with serines, the 18 consecutive residues from S184 to S201 which are believed to form the transmembrane segment IV were substituted individually with cysteine and labeled with a thiol-selective nitroxide reagent. Most of the labeled mutants exhibited significant oxoglutarate transport in reconstituted liposomes, where they were examined by EPR as a function of the incident microwave power in the presence and absence of two paramagnetic perturbants, i.e., the hydrophobic molecular oxygen or the hydrophilic chromium oxalate complex. The periodicity of the sequence-specific variation in the spin-label mobility and the O2 accessibility parameters unambiguously identifies the fourth transmembrane segment of the mitochondrial oxoglutarate carrier as an α-helix. The accessibility to chromium oxalate is out of phase with oxygen accessibility, indicating that the helix is amphipatic, with the hydrophilic face containing the residues found to be important for transport activity by site-directed mutagenesis and chemical modification. The helix is strongly packed, as indicated by the values of normalized mobility, which also suggest that the conformational changes occurring during transport probably involve the N-terminal region of the helix.
2003
Pubblicato
Rilevanza internazionale
Articolo
Sì, ma tipo non specificato
Settore BIO/10 - BIOCHIMICA
English
Con Impact Factor ISI
Oxoglutaric acid; carrier protein; liposome; nitroxide; dynamics; electron spin resonance; mitochondria; mutagenesis; spin labeling; ketoglutaric acids; membrane transport proteins; site-directed
MOROZZO DELLA ROCCA, B., Lauria, G., Venerini, F., Palmieri, L., Polizio, F., Capobianco, L., et al. (2003). The mitochondrial oxoglutarate carrier: Structural and dynamic properties of transmembrane segment IV studied by site-directed spin labeling. BIOCHEMISTRY, 42, 5493-5499 [10.1021/bi027025q].
MOROZZO DELLA ROCCA, B; Lauria, G; Venerini, F; Palmieri, L; Polizio, F; Capobianco, L; Stipani, V; Pedersen, Jz; Cappello, A; Desideri, A; Palmieri, ...espandi
Articolo su rivista
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2108/44607
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 15
  • ???jsp.display-item.citation.isi??? 14
social impact