Thermal unfolding of the PDZ domain of β2-sintrophin followed by RMN and CD spectroscopy

GABRIELA MARÍA TORCHIO; MARIANA GALLO; MARIO ROBERTO ERMÁCORA; MAURICIO PABLO SICA

San Javier

Congreso; XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

P { margin-bottom: 0.21cm; direction: ltr; color: rgb(0, 0, 0); text-align: left; }P.western { font-family: "Times New Roman",serif; font-size: 12pt; }P.cjk { font-family: "Droid Sans"; font-size: 12pt; }P.ctl { font-family: "Lohit Hindi"; font-size: 12pt; } PDZ domains are protein-protein interaction modules found in almost every kingdom of life. β2-sintrophin (B2S) is a cytoplasmatic protein found in the beta cells from pancreas, and plays a key role in anchoring insulin containing secretion granules (SG) to the actin cytoskeleton (1). In this model, B2S interacts through its PDZ domain with IA-2, a trasnmembrane protein located in the membrane of the SG. Upon insulin secretion stimulation, IA-2 and B2S dissociate from each other, and the SG is mobilized to the plasmatic membrane, where the insulin is released to the extracellular space by exocitosys (2,3). Previously we showed interesting results from thermal denaturation experiments of the PDZ domain of B2S (B2S-PDZ). It was found that B2S-PDZ is less stable than other reported PDZ domains, and that it has some particular unfolding characteristics. Thermal unfolding experiments at different urea concentrations, followed by circular dichroism (CD), revealed that B2S-PDZ does not unfolds as expected for two-state or even three-state models. In fact, the unfolding curves obtainde are more reminiscent of those of a protein that follows a downhill unfolding model (4,5). Here we present a preliminary characterization of B2S-PDZ by one dimension 1H nuclear magnetic resonance (1D-NMR). The hydrodinamic radius (Rh) determined by this technique was 17.1 +/- 0.3 A, measured at 20 °C, as expected for a globular protein of this size (6). High-field region of 1D-NMR spectra remain structured until 320K, compatible with the unfolding transition observed by circular dichroism. However, low-field region shows a complex dependence with temperature. Our results reinforce the idea the folding mechanism of B2S-PDZ cannot be explained satisfactorily by a discrete-states model.