DSC and RMN approach to study the complex unfolding mechanism of a pdz domain

TORCHIO G; BURGOS I; FIDELIO G; ARÁN M; GALLO M; ERMACORA MR; SICA M

Villa Carlos Paz, Córdoba

Congreso; Sociedad Argentina de Biofísica XLII Reunión Annual; 2013

SAB

Many PDZ domains have been extensively studied from a biophysical perspective since they are important and ubiquitous modules of protein-protein interactions, with particular characteristics of folding and binding. Our model of study is the PDZ domain of the β2-syntrophin protein (β2S-PDZ), which is directly involved in the regulation of insulin secretion. We have shown that thermal unfolding curves of β2S-PDZ are not as expected for a protein that follows a two state nor a three state model of folding (1), as reported for other PDZ domains. Moreover, the unfolding curves of β2S-PDZ are more reminiscent of a downhill regime, or multiple state mechanism of folding (2-3). Here we present additional evidence that supports the hypothesis of a complex folding mechanisms for β2S-PDZ. Differential scanning calorimetry (DSC) experiments discard two- and three-state models, showing a more complex scenario. DSC experiments also rule out oligomerization during thermal unfolding, at least at concentrations up to 2 mg/ml. Although some aggregation is detected at high temperatures, this only seems to be associated to a minor population of β2S-PDZ and thermal denaturation probed to be highly reversible, as a second and a third scan of DSC are completely superimposable. Also NMR experiments suggest the existence of a minor population of β2S-PDZ that could populate an alternative conformation.