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Detection and Characterization of Partially Unfolded Oligomers of the SH3 Domain of -Spectrin

Salvador Casares ^*, Mourad Sadqi ^*, Obdulio López-Mayorga ^*, Francisco Conejero-Lara ^* and Nico A. J. van Nuland 

^* Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain; and Bijvoet Centre, Department of NMR Spectroscopy, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands

Correspondence: Address reprint requests to Francisco Conejero-Lara, Tel.: +34-958-242371; Fax: +34-958-272879; E-mail: conejero{at}ugr.es.

For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of -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 0.5 mM the population of the intermediate state becomes significant at temperatures between 45°C and 80°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.

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