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Detection and characterization of partially unfolded oligomers of the SH3 domain of alpha-spectrin.
- Source :
-
Biophysical journal [Biophys J] 2004 Apr; Vol. 86 (4), pp. 2403-13. - Publication Year :
- 2004
-
Abstract
- For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of alpha-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning calorimetry, circular dichroism, and NMR follow apparently the two-state model when each unfolding profile is considered individually. Nevertheless, we have found that protein concentration has a marked effect upon the thermal unfolding profiles. This effect cannot be properly explained in terms of the two-state unfolding model and can only be interpreted in terms of the accumulation of intermediate associated states in equilibrium with the monomeric native and unfolded states. By chemical cross-linking and pulsed-field gradient NMR diffusion experiments we have been able to confirm the existence of associated states formed during spc-SH3 unfolding. A three-state model, in which a dimeric intermediate state is assumed to be significantly populated, provides the simplest interpretation of the whole set of thermal unfolding data and affords a satisfactory explanation for the concentration effects observed. Whereas at low concentrations the population of the associated intermediate state is negligible and the unfolding process consequently takes place in a two-state fashion, at concentrations above approximately 0.5 mM the population of the intermediate state becomes significant at temperatures between 45 degrees C and 80 degrees C and reaches up to 50% at the largest concentration investigated. The thermodynamic properties of the intermediate state implied by this analysis fall in between those of the unfolded state and the native ones, indicating a considerably disordered conformation, which appears to be stabilized by oligomerization.
Details
- Language :
- English
- ISSN :
- 0006-3495
- Volume :
- 86
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Biophysical journal
- Publication Type :
- Academic Journal
- Accession number :
- 15041678
- Full Text :
- https://doi.org/10.1016/S0006-3495(04)74297-6