Equilibrium denaturation study of the mature ectodomain of the protein-tyrosine phosphatase IA-2.

NOGUERA. MMEE; M.E. PRIMO; SOSA, L.N.F; RISSO,V.A; ERMÁCORA, M.R.

Buzios

Congreso; VII Congreso Iberoamericano de Biofísica.; 2009

Sociedad Iberoamericana de Biofísica. Sociedad Argentina de Biofísica

Introduction IA-2 is a protein tyrosine phosphatase receptor located in secretory vesicles. Posttraslational modifications remove the signal peptide and an adjacent fragment. Mature IA-2 comprises an ectodomain (residues 449-575), a single pass transmembrane region (576-600) and a cytoplasmatic domain (601-979). The mature ectodomain (meIA-2) is self-proteolyzed in vitro by reactive oxygen species, yielding a fragment that comprises the residues 469-557 of IA-2. X-ray structure of meIA-2 revealed a homodimeric structure, with two possible association modes between monomers (1). Dimerization might  to play a functional role in mediating interactions with molecules of the extracellular matrix and membranes from different cells. Objective The objective of this study was to determine the conformational stability of meIA-2 and the relative contribution of quaternary interactions to the global stability of the dimer. The protein fragment comprising residues 469 to 557 of human IA-2 was expressed in soluble form in E. coli, and purified by a three-step ionic exchange chromatography protocol. Circular dichroism (CD) spectra in the far-UV region were recorded on a Jasco J-810 spectropolarimeter using 0.1 cm quartz cuvettes. Fluorescence measurements were recorded using an ISS K-2 spectrofluorometer. The fluorescence emission was collected through a WG320 filter while the excitation wavelength was set at 276 nm. The unfolding of meIA-2 by guanidinium chloride (GdnCl) was followed by fluorescence and circular dichroism. Separate samples of meIA-2 were incubated >4h at room temperature in presence of increasing amounts of denaturant. All measurements were carried out at 20°C. The denaturation curves were analized according to a two and three-state unfolding models, using the combined CD and fluorescence data. Results The denaturation is reversible and cooperative. The unfolding is protein concentration-dependent and apparently monophasic. Global analysis indicates that the data are better described by fitting to a three-state model. Although the statistical analysis do not allow to discard one of the three-state models, the mechanism involving a monomeric intermediate is the most consistent with the denaturation data. meIA-2 exhibit a high thermodynamic stability. The free energy change involved in dissociation process is greater than the change involved in unimolecular unfolding event. Conclusion We have determined that the quaternary structure of meIA-2 provides a major contribution to the high conformational stability of this protein. The total m value for the transition is the expected from the crystallographic structure, regardless of the fitting model. Further experiments are underway to establish the properties of the intermediate. References (1) Primo, M.E. et al, J. Biol. Chem. 2008, 283, 4674-81.