Thermal unfolding of small proteins with SH3 domain folding pattern

Abstract

The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy. The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 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 heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities.

Phylogeny of Tec Family Kinases: Identification of a Premetazoan Origin of Btk, Bmx, Itk, Tec, Txk, and the Btk Regulator SH3BP5

“…The thermal unfolding pattern of different TFK SH3 domains has also been analyzed (Knapp et al, 1998). In contrast to most other SH3 domains, the TFK family seems to be regulated by tyrosine phosphorylation as demonstrated for Btk (Park et al, 1996) and Itk (Hao and August, 2002;Wilcox and Berg, 2003) as well as Tec and Bmx (Nore et al, 2003).…”

Bruton tyrosine kinase (BTK) in X-linked agammaglobulinemia (XLA)

“…The Btk has the typical SH3 domain topology of two short anti-parallel ß-sheets packed almost perpendicular to each other in a sandwich-like fold. Thermal unfolding of Tec family SH3 domains have been studied with CD spectroscopy and isothermal titration calorimetry (128).…”

Dynamics of the Tec‐family tyrosine kinase SH3 domains

“…Despite apparent similarities in tertiary structure, SH3 domain stability and dynamics vary widely. 10 For example, the Drk N-terminal SH3 domain, which has been studied extensively by NMR, was found to exist in equilibrium between a folded and unfolded state under non-denaturing conditions at ratio of 2:1 with an exchange rate constant of 2.2 s 21 . 11 Previously, our group probed the solution dynamics of a number of SH3 domains with hydrogen exchange mass spectrometry (HX MS) and found unpredictable dynamics and partial unfolding at near-physiological conditions.…”