Determination of the hydrophobic surface of P-ATPases using a photoactivatable labeled probe.

SAFFIOTI, NICOLÁS ANDRÉS; MANGIALAVORI, IRENE CECILIA; BERLIN, JOSHUA; ROSSI, JUAN PABLO

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

Sociedad Argentina de Biofisica - Seccional Biofísica de la Sociedad Uruguaya de Biociencias

The detailed P-ATPases mechanism is a question that it still remains unanswered. The X-ray crystallography models of the sarcoplasmic reticulum calcium pump (SERCA) and the sodium potassium ATPase, have given information about the structure of these proteins in different steps of their reaction cycle1. However since membrane proteins are very difficult to crystallize, this technique has only been successful in few P-ATPases and in certain conditions. In order to study structure of membrane proteins, we developed a method using a hydrophobic photoactivatable probe, which under UV irradiation is capable to bind covalently to the macromolecule. This property allows us to label the exposed surface to the lipid bilayer and hydrophobic pockets in P-ATPases. In this work we design a novel precursor of the 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) easy to be labeled with 125I. Once obtained the probe, we characterize the photolabeling in different members of the family and studied them under different conditions. We found that SERCA and plasma membrane calcium pump label less when both proteins are in E1 conformation. This result agrees with a less exposed surface to the bilayer as seen in the crystallographic structure of SERCA2. Further, we tested the effect of different flavonoids in the SERCA and found that its binding elicits a conformation higher exposed to the hydrophobic milieu.[1] Toyoshima C, et al. Nature 418:605-11, 2002.[2] Mangialavori I, et al. J Biol Chem 284:4823-8, 2009.With grants of ANPCYT, UBACYT and CONICET.