Navegando por Autor "Abiko, Layara Akemi"

Filtrar resultados informando as primeiras letras
Agora exibindo 1 - 1 de 1
  • Nenhuma Miniatura Disponível
    Item
    CALX-CBD1 Ca2D-binding cooperativity studied by NMR spectroscopy and ITC with bayesian statistics.
    (2020) Cardoso, Marcus Vinícius Cangussu; Rivera, José Domingo; Vitale, Phelipe Augusto Mariano; Degenhardt, Maximilia Frazão de Souza; Abiko, Layara Akemi; Oliveira, Cristiano Luis Pinto de; Salinas, Roberto Kopke
    The Naþ/Ca2þ exchanger of Drosophila melanogaster, CALX, is the main Ca2þ-extrusion mechanism in olfactory sensory neurons and photoreceptor cells. Naþ/Ca2þ exchangers have two Ca2þ sensor domains, CBD1 and CBD2. In contrast to the mammalian homologs, CALX is inhibited by Ca2þ binding to CALX-CBD1, whereas CALX-CBD2 does not bind Ca2þ at physiological concentrations. CALX-CBD1 consists of a b-sandwich and displays four Ca2þ-binding sites at the tip of the domain. In this study, we used NMR spectroscopy and isothermal titration calorimetry (ITC) to investigate the cooperativity of Ca2þ bind- ing to CALX-CBD1. We observed that this domain binds Ca2þ in the slow exchange regime at the NMR chemical shift timescale. Ca2þ binding restricts the dynamics in the Ca2þ-binding region. Experiments of 15N chemical exchange saturation transfer and 15N R2 dispersion allowed the determination of Ca2þ dissociation rates (30 s1 ). NMR titration curves of residues in the Ca2þ- binding region were sigmoidal because of the contribution of chemical exchange to transverse magnetization relaxation rates, R2. Hence, a novel, to our knowledge, approach to analyze NMR titration curves was proposed. Ca2þ-binding cooperativity was examined assuming two different stoichiometric binding models and using a Bayesian approach for data analysis. Fittings of NMR and ITC binding curves to the Hill model yielded nHill 2.9, near maximal cooperativity (nHill 1⁄4 4). By assuming a stepwise model to interpret the ITC data, we found that the probability of binding from 2 up to 4 Ca2þ is approximately three orders of magnitude higher than that of binding a single Ca2þ. Hence, four Ca2þ ions bind almost simultaneously to CALX-CBD1. Coop- erative Ca2þ binding is key to enable this exchanger to efficiently respond to changes in the intracellular Ca2þ concentration in sensory neuronal cells.