El desafío de las proteínas de membrana: Desandando los cambios conformacionales que permiten el transporte de calcio

SAFFIOTI, NICOLÁS ANDRÉS; DE SAUTU, MARILINA; ROSSI ROLANDO; BERLIN, JOSHUA; ROSSI, JUAN PABLO; FERRIERA GOMES, MARIELA; MANGIALAVORI, IRENE CECILIA

Capital federal

Congreso; II Simposio de jóvenes científicos; 2017

The challenge of membrane protein study: Unravelling the structural changes that allow ion transport in the plasma membrane calcium pump.Saffioti Nicolás Andrés1, de Sautu Marilina1, Rossi Rolando1, Berlin Joshua2, Rossi Juan Pablo1, Ferreira-Gomes Mariela1, Mangialavori Irene11Departamento de Química Biológica, IQUIFIB, Capital Federal, Buenos Aires, Argentina2Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers University, Newark, NJ, USA.The study of membrane proteins presents important hindrances compared with soluble proteins. Expression and purification are harder and often techniques for studying structure are limited, especially in the case of big proteins. Plasma Membrane Calcium Pump (PMCA) is a protein of this kind. It is a member of P-ATPase family, very important for calcium handling and present in all Eukaryotes. We want to understand how the structure of this protein changes during the catalytic cycle, and for this we are employing different inhibitors. Particularly, fluoride compounds stabilize P-ATPases in different analogues of the phosphorylated state but they have never been assayed on PMCA.Our results show that fluoride complexes of beryllium, magnesium and aluminum inhibit PMCA transport activity by preventing phosphorylation of the pump. We studied the transmembrane region using photoactivatable probes showing that this part of the protein has flexibility even when the pump has bound fluoride complexes. We also employed fluorescent probes to study the behavior of the N-domain. Quantum yield of the probes bound to the protein decreased upon complexes binding, indicating a more exposure to solvent at this domain in the phosphorylated state. The magnitude of this change depends of the complex assayed and can be followed as function of time. This allows us to propose a kinetic model for explaining the effect of each fluoride complex on the pump.Finally, we employed the structural information presented above to assess different structural models of PMCA, constructed by homology with Sarcoplasmic Reticulum Calcium Pump (SERCA) and the Sodium Potassium ATPase (Na+/K+ ATPase). This work was supported by Agencia Nacional de Promoción Científica y Tecnológica PICT 2014 0065, Consejo Nacional de Investigaciones Científicas y Técnicas PIP 11220150100250CO, and Universidad de Buenos Aires Ciencia y Técnica grant 2014-2017: 20020130100254B