Special folding pathway to tetramer only through the micelle state of the corticotropin-releasing factor

The ovine corticotropin-releasing factor (CRF), a peptide hormone of 41 residues stimulating the secretion of adrenocorticotropic hormone, was thermodynamically investigated. By means of size exclusion chromatography and/or ultrafiltration, the CRF solution could be separated into random coil monomers and highly alpha-helical tetramers, which seem to have amphipathic helix bundle structure. Circular dichroism measurements along with diluting or concentrating the CRF solution revealed that there exists the micelle state above the concentration of 0.1 mM, which would be the critical micelle concentration (cmc). The micelle state was also proved by binding ability for 8-anilino-1-naphthalenesulfonate and endothermic change by dilution across the cmc. The tetramer showed the cooperative thermal transition at about 55 degrees C in the buffer solution (pH 7.5), so that it would have native-protein-like folding. On the other hand, the micelle undergoes gradual change to dissociated state by heating, regardless of the similar alpha-helicity to the tetramer. Above the cmc the equilibrium between the tetramer and the micelle takes place as well as that between the monomer and the micelle. Whereas, the direct conversion between the tetramer and the monomer scarcely occurred below the cmc. The titration experiment with 2,2,2-trifluoroethanol (TFE) revealed that the cmc decreases with increasing the concentration of TFE. This tendency is the same as that of general surfactants. Most of experimental results can be well explained by this three-phase model involving the monomer, the tetramer, and the micelle. The lack of the equilibrium between the monomer and the tetramer indicates that the folding pathway of the tetramer is the transformation only through the micelle state and not from the monomer. This pathway resembles the collapse model among the phenomenological models for thermodynamic protein folding. By the mathematical consideration for the dissociation of micelle, we have demonstrated that the expected content of undegradable k-mer is 2/(k + 1), which agreed well with the observed tetramer content of CRF (40%).