THERMODYNAMIC AND KINETIC-ANALYSIS OF THE SH3 DOMAIN OF SPECTRIN SHOWS A 2-STATE FOLDING TRANSITION

The folding and unfolding reactions of the SH3 domain of spectrin can be described by a two-state model. This domain is a beta-sheet barrel containing 62 amino acids. Equilibrium unfolding by urea, guanidine hydrochloride, and heat is completely reversible at pH values below 4.0. At higher pH values the unfolding is reversible as long as the protein concentration is below 1 mg/mL. The Gibbs energy of unfolding in the absence of denaturant, Delta G(H2O), at pH 3.5 and 298 K is calculated to be 12 kJ mol(-1) for urea, chemical, and temperature denaturation. The stability of the protein does not change noticeably between pH 5.0 and 7.0 and is around 15.5 kJ mol(-1). Since heat effects of unfolding are relatively small and, as a result, heat-induced melting occurs in a wide temperature range, the analysis of scanning calorimetry data was performed taking into account the temperature dependence of unfolding Delta C-p,. The free energy of unfolding obtained for this domain (Delta G(H2O) 14 +/- 2 kJ mol(-1)) was, within experimental error, similar to those obtained in this work by other techniques and with those reported in the literature for small globular proteins. Kinetics of unfolding and refolding at pH 3.5, followed both by fluorescence and by circular dichroism, provide evidence of the simplest folding mechanism consistent with the two-state approximation. A value for Delta G(H2O) = 13 +/- 0.7 kJ mol(-1) can be extrapolated from the kinetic data. No intermediate can be seen to accumulate by equilibrium denaturation followed by fluorescence and circular dichroism, refolding kinetics and calorimetry, and a concomitant recovery of secondary and teritary structure is observed during refolding. This suggests that the two-state model can properly describe the folding of this domain from both the equilibrium and kinetic points of view and raises the question of whether the accumulation of kinetic intermediates is merely a result of the size of the protein being studied.