title: Substrate-induced conformational changes of the mitochondrial oxoglutarate carrier: a spectroscopic and molecular modelling study.
abstract: The structural and dynamic properties of the oxoglutarate carrier were investigated by introducing a single tryptophan in the Trp-devoid carrier in position 184, 190 or 199 and by monitoring the fluorescence spectra in the presence and absence of the substrate oxoglutarate. In the absence of substrate, the emission maxima of Arg190Trp, Cys184Trp and Leu199Trp are centered at 342, 345 and 348 nm, respectively, indicating that these residues have an increasing degree of solvent exposure. The emission intensity of the Arg190Trp and Cys184Trp mutants is higher than that of Leu199Trp. Addition of substrate increases the emission intensity of Leu199Trp, but not that of Cys184Trp and Arg190Trp. A 3D model of the oxoglutarate carrier was built using the structure of the ADP/ATP carrier as a template and was validated with the experimental results available in the literature. The model identifies Lys122 as the most likely candidate for the quenching of Trp199. Consistently, the double mutant Lys122Ala-Leu199Trp exhibits a higher emission intensity than Leu199Trp and does not display further fluorescence enhancement in response to substrate addition. Substitution of Lys122 with Cys and evaluation of its reactivity with a sulphydryl reagent in the presence and absence of substrate confirms that residue 122 is masked by the substrate, likely through a substrate-induced conformational change.
authors.affiliation: INFM and Department of Biology, University of Rome "Tor Vergata", via della Ricerca Scientifica, Rome.
authors.forename: Blasco
authors.initials: B
authors.lastname: Morozzo Della Rocca
authors.forename: Daniela V
authors.initials: DV
authors.lastname: Miniero
authors.forename: Gianluca
authors.initials: G
authors.lastname: Tasco
authors.forename: Vincenza
authors.initials: V
authors.lastname: Dolce
authors.forename: Mattia
authors.initials: M
authors.lastname: Falconi
authors.forename: Anna
authors.initials: A
authors.lastname: Ludovico
authors.forename: Anna R
authors.initials: AR
authors.lastname: Cappello
authors.forename: Paloma
authors.initials: P
authors.lastname: Sanchez
authors.forename: Italo
authors.initials: I
authors.lastname: Stipani
authors.forename: Rita
authors.initials: R
authors.lastname: Casadio
authors.forename: Alessandro
authors.initials: A
authors.lastname: Desideri
authors.forename: Ferdinando
authors.initials: F
authors.lastname: Palmieri
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007656:Ketoglutaric Acids / Q000737:chemistry; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
chemical_list: D007656:Ketoglutaric Acids; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; C021723:oxoglutarate translocator; D014443:Tyrosine; D014364:Tryptophan; D008239:Lysine; D003545:Cysteine