Transmembrane domains of IA-2

PRIMO MARÍA EVANGELINA; RISSO VALERIA A.; SOSA LAURA N.F.; NOGUERA MARTÍN EZEQUIEL; POSKUS EDGARDO; ERMÁCORA MARIO ROBERTO

Buenos Aires

Jornada; Jornada "Herramientas Modernas para el estudio de aspectos estructurales de proteinas; 2009

Academia Nacional de Farmacia y Bioquímica

Protein-tyrosine phosphatase receptor IA-2 is post translationally processed by proteolytic cleavage, yielding a mature extracellular domain (meIA-2, residues 449-576), attached to the naturally inactive intracellular domain through a single transmembrane region (residues 576-600). In a previous work we reported the X-ray structure of two dimerizacion forms of meIA-2 and characterized its free radical mediated oxidative degradation to yield meIA-2 comprising residues 469-557. The aim of the present work was to prepare, purify and biophysically characterize the me-tmIA-2 tandem (residues 449-600) in a folded state. His-tag me-tmIA-2 was expressed in E. coli and purified by metal affinity chromatography in buffers containing 0.1% Thesit. meIA2 was prepared according to our published protocol (Primo et al. 2008, JBC). Far- and near-UV spectroscopy measurements were carried out at 20ºC and at pH 7.0. Crosslinking was performed with DSS. Limited proteolysis was carried out with trypsin (100:1, w/w, protein/protease ratio) at 28°C, with or without 2 M urea. The prepared proteins were >95% pure. His-tag me-tmIA2 bound to detergent micelles exhibited near and far UV-CD spectra consistent with a native fold, as judged by comparative analysis of meIA2 under identical conditions. Both proteins were similarly resistant to trypsin. Cross-linking experiments evidenced that both protein dimerize in solution. A protocol for the production of folded me-tmIA2 was devised and successfully tested. me-tmIA2 exhibits spectral features similar to those of meIA-2. These findings open the way for higher resolution studies aimed to establish the disposition and insertion mode of the extracellular domain of IA2 in artificial and natural membranes.