Folding, stability, and secondary structure of a new dimeric cysteine proteinase inhibitor

Clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, is a 34-kDa homodimer lacking disulphide bonds, reported to have unusual stability properties. Sequence similarity is limited solely to certain proteins from mushrooms. Infrared spectroscopy shows that clitocypin is a high β-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical β-structure, but similar to those of a group of small β-strand proteins, with a peak at 189 nm and a trough at 202 nm. An aromatic peak at 232 nm and infrared bands at 1633 and 1515 cm −1 associated with the peptide backbone and the tyrosine microenvironment, respectively, were used to characterize the thermal unfolding. The reversible transition has a midpoint at 67 °C, with ΔG=34 kJ/mol and ΔH=300 kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44 kJ/mol, respectively. A model for folding and assembly is discussed.