Thermal unfolding of the PDZ domain of beta2- syntrophin characterized by NMR

GABRIELA TORCHIO, MARTÍN ARÁN, MARIANA GALLO, MARIO ROBERTO ERMÁCORA Y MAURICIO SICA

Sierra de la Ventana, Buenos Aires

Congreso; XLIII Reunión Anual de SAB; 2014

SAB

Beta2 syntrophin regulates insulin interactions of its PDZ domain (B2S-PDZ). Thermal unfoling of B2S-PDZ do not fit properly to models with discrete (two- or three) states, and may involve low or negligible barriers. In this case, known as downhill unfolding, the protein populates a unique state whose conformational characteristics varies with temperature, and thus several structure element can unfold (quasi) independently. Our previous studies indicate that B2S-PDZ unfolfing better fits to low barriers models. To get a deeper, we applied NMR spectroscopy the unfolding of B2S-PDZ. For this purpose, 15N-1H-HSQC of B2S-PDZ were obtained at different  temperatures, from 5 to 50 °C. T1, T2 and NOE at 20°C indicate that the protein is monomeric and rigid and except for helix A that suffer slow conformational movements (0.1- 1 ms). Since the intensity of the signals do not decrease with the temperature, the chemical shift variations are consider to occur in a fast exchange regime. The highest temperatures allowed by the technique corresponded to Tm from far-UV Cd and DSC experiments. Thus, after assigning each the spectra signals the experiment gave information about the early conformational behavior, showing that several elements suffer a gradual thermal unfolding. These elements involve beta strands D,F, both ends of helix B and C-end of beta strand C. These elements are clusterd in a region that harbor several residues involved in the PDZ-ligand binding. Our results indicate that B2S-PDZ is gradually unfolded at least at temperatures previous to apparent Tm, in agreement with a downhill model. Furthermore, this behavior could be of relevance to understand the regulation of the  function of this domain.