IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Study of ligand binding sites in Plasma Membrane Calcium Pump: An Azido-Ruthenium photoreactive probe as strategy for structural characterization
Autor/es:
MANGIALAVORI IRENE; ROSSI JEAN PAUL; GENTILE LUCILA; ERRA BALSELLS ROSA; ONTIVEROS MALLKU; PETROSELLI GABRIELA; FERREIRA-GOMES MARIELA
Lugar:
San Miguel de Tucumán
Reunión:
Congreso; III Latin American Federation of Biophysical Societies (LAFeBS) / IX IberoAmerican Congress of Biophysics / XLV Reunión Anual SAB 2016; 2016
Institución organizadora:
SAB
Resumen:
The Plasma Membrane Calcium ATPase (PMCA) is a P-type ATPase that maintains the homeostasis of Ca2+ in eukaryotic cells. It couples the transport of Ca2+ with the hydrolysis of ATP. The structure of PMCA is still not resolved, and only limited information is available of ligand binding sites. The purpose of this work is to identify and characterize the ligand binding sites of PMCA. We synthesized azido-ruthenium (AzRu) a photoactivable reagent to obtain structural information of PMCA, which binds covalently and specifically to Ca2+-binding proteins after exposure to irradiation at 290 nm1. The experiments were performed with purified PMCA from human erythrocytes. Measurements of phosphoenzyme in presence of AzRu showed an increase of phosphorylated intermediate in experimental conditions that inhibit the ATPase activity. This suggests that AzRu would be affecting the dephosphorylation of the pump.Studies of ESI-Orbitrap/MALDI-TOF-MS of PMCA with AzRu (after photolysis) showed that some peptides cannot be found, suggesting that these peptides might be related with the interaction sites between AzRu and PMCA. These peptides contain histidine residues and other residues that are targets of covalent binding with the photoactivable azido group2. We analyzed the sequence homology model of PMCA on crystallographic structure of SERCA and we found a peptide related with the Mg2+ site. Other peptides were analyzed because these are in the C-terminus which is not present in SERCA. Ours results suggest that the Mg2+ binding site could be involved in the interaction between PMCA and AzRu.We analyzed the structure of AzRu by MALDI/LDI-TOF and found fragments of reagent that would be photolysis products. This result will be useful to recognize adducts AzRu-PMCA and easily analysis of the results by mass spectrometry.