THERMAL UNFOLDING OF SMALL PROTEINS WITH SH3 DOMAIN FOLDING PATTERN

The thermal unfolding of three SH3 domains of the Tec family of tyrosi ne kinases was studied by differential scanning calorimetry and CD spe ctroscopy, The unfolding transition of the three protein domains in th e acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH re gion 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec), At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk- , Itk-, and Tec-SH3 domains, respectively. The determined changes in h eat capacity between the native and the denatured state are in an usua l range expected for small proteins. Our analysis revealed that all SH 3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high meltin g temperatures. Comparing unfolding free energies measured for eukaryo tic SH3 domains with those of the topologically identical Sso7d protei n from the hyperthermophile Sulfolobus solfataricus, the increased mel ting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at the ir physiological temperatures, 310K for mesophilic SH3 domains and 350 K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabil ities. (C) 1998 Wiley-Liss, Inc.