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

SAFFIOTI, N., BERLIN J., ROSSI J.P.AND AND MANGIALAVORI, IC

Salto

Congreso; Latin American crosstalk in Biophysics and physiology.; 2015

Determination of the hydrophobic surface of P-ATPases using a photoactivatable labeled probe. SBF.uy/SAB 2015 Saffioti Nicolás Andrés1,Mangialavori Irene Cecilia1, Berlin Joshua 2, Rossi JuanPablo1,  1IQUIFIB-Departamento de Química Biológica, Facultad de Farmacia yBioquímica, UBA-CONICET, 2 New JerseyMedical School, Rutgers UniversityCorresponding author: nsaffioti@gmail.comThedetailed 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 thestructure of these proteins in different steps of their reaction cycle1.However since membrane proteins are very difficult to crystallize, thistechnique 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 ahydrophobic photoactivatable probe, which under UV irradiation is capable tobind covalently to the macromolecule.  Thisproperty allows us to label the exposed surface to the lipid bilayer andhydrophobic pockets in P-ATPases. In this work we design a novel precursor ofthe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) easy to be labeled with125I. Once obtained the probe, we characterize the photolabeling indifferent members of the family and studied them under different conditions. Wefound that SERCA and plasma membrane calcium pump label less when both proteinsare in E1 conformation. This result agrees with a less exposedsurface to the bilayer as seen in the crystallographic structure of SERCA2.Further, we tested the effect of different flavonoids in the SERCA and foundthat its binding elicits a conformation higher exposed to the hydrophobicmilieu.[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.