title: Detection and characterization of partially unfolded oligomers of the SH3 domain of alpha-spectrin.
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.
authors.affiliation: Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain.
authors.forename: Salvador
authors.initials: S
authors.lastname: Casares
authors.forename: Mourad
authors.initials: M
authors.lastname: Sadqi
authors.forename: Obdulio
authors.initials: O
authors.lastname: López-Mayorga
authors.forename: Francisco
authors.initials: F
authors.lastname: Conejero-Lara
authors.forename: Nico A J
authors.initials: NA
authors.lastname: van Nuland
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002645:Chickens; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D013049:Spectrin / Q000737:chemistry*; D013816:Thermodynamics; D018909:src Homology Domains
chemical_list: D003432:Cross-Linking Reagents; D013049:Spectrin