Thermodynamic and kinetic analysis of the SH3 domain of spectrin shows a two-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 GH2O, 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 Cp. The free energy of unfolding obtained for this domain (delta GH2O = 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 GH2O = 13 +/- 0.7 kJ mol-1 can be extrapolated from the kinetic data.(ABSTRACT TRUNCATED AT 250 WORDS)
Viguera
A R
AR
European Molecular Biology Laboratory, Heidelberg, Germany.
Martínez
J C
JC
Filimonov
V V
VV
Mateo
P L
PL
Serrano
L
L
D001665:Binding Sites; D002942:Circular Dichroism; D006358:Hot Temperature; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry; D013049:Spectrin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D013049:Spectrin; D016392:Proto-Oncogene Proteins pp60(c-src)