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 hy drochloride, and heat is completely reversible at pH values below 4.0. At higher pH values the unfolding is reversible as long as the protei n 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 calculate d 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 unfold ing are relatively small and, as a result, heat-induced melting occurs in a wide temperature range, the analysis of scanning calorimetry dat a was performed taking into account the temperature dependence of unfo lding Delta C-p,. The free energy of unfolding obtained for this domai n (Delta G(H2O) 14 +/- 2 kJ mol(-1)) was, within experimental error, s imilar to those obtained in this work by other techniques and with tho se 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 mechan ism consistent with the two-state approximation. A value for Delta G(H 2O) = 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 a nd calorimetry, and a concomitant recovery of secondary and teritary s tructure is observed during refolding. This suggests that the two-stat e model can properly describe the folding of this domain from both the equilibrium and kinetic points of view and raises the question of whe ther the accumulation of kinetic intermediates is merely a result of t he size of the protein being studied.