IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
STRUCTURE OF DIFFERENT PHOSPHORYLATED STATES OF PLASMA MEMBRANE CALCIUM PUMP
Autor/es:
SAFFIOTI NICOLÁS; BERLIN, JOSHUA; MANGIALAVORI IRENE; ROSSI ROLANDO; FERREIRA GOMES, MARIELA; DE SAUTU, MARILINA; ROSSI JPFC
Lugar:
Buenos Aires
Reunión:
Congreso; REUNIÓN CONJUNTA DE SOCIEDADES DE BIOCIENCIAS; 2017
Resumen:
The control of the cytoplasmic calcium homeostasis relies on manytransporters like the Plasma Membrane Calcium Pump (PMCA) andthe Sarcoplasmic Reticulum Calcium Pump (SERCA) which belongto the P-ATPase family. At difference with SERCA, structure andfunction of PMCA has not been fully elucidated yet, because obtainingsuitable preparations for X-ray crystal diffraction and NMRtechniques was unsuccessful.To elucidate the structural changes produced in PMCA during thereaction cycle we tested a purified preparation of the pump withfluoride complexes of beryllium, aluminum and magnesium, eachone stabilizing different analogues of phosphorylated intermediatesin P-ATPases. These blockers were previously assayed in SERCAleading to different conformational states. To follow the binding offluoride complexes by fluorescence we employed eosin, which bindsto the N domain of PMCA. Quantum yield of the bound probe decreasedin the presence of fluoride complexes indicating more exposureto solvent. The magnitude of this change depended on thepresence of calcium and the complex identity.Following the kinetics of the conformational change, we proposea model to explain how these complexes stabilize phosphorylatedstates in PMCA. The detectable conformational change associatedwith an intermediate of the reaction cycle also allows us to developa method to measure PMCA activity using a fluorescence approach.Finally, we prepared and refined PMCA structural models based onhomology with SERCA and Na+/K+ ATPase at different states, to understandthe changes at the nucleotide-binding domain during thecatalytic cycle of PMCA.This work was supported by Agencia Nacional de PromociónCientífica y Tecnológica PICT 2014 0065, Consejo Nacional de InvestigacionesCientíficas y Técnicas PIP 11220150100250CO, andUniversidad de Buenos Aires Ciencia y Técnica grant 2014-2017:20020130100254B