The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan inthe Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absenceof the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trpare centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solventexposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition ofsubstrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of theoxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with theexperimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenchingof Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trpand does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cysand evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122is masked by the substrate, likely through a substrate-induced conformational change.
Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study
DOLCE V;CAPPELLO AR;
2005-01-01
Abstract
The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan inthe Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absenceof the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trpare centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solventexposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition ofsubstrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of theoxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with theexperimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenchingof Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trpand does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cysand evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122is masked by the substrate, likely through a substrate-induced conformational change.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
