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 beta-structure protein which was lost at high temperatures. The far UV circular dichroism spectrum is not that of classical beta-structure, but similar to those of a group of small beta-strand proteins, with a peak at 189nm and a trough at 202nm. An aromatic peak at 232nm and infrared bands at 1633 and 1515cm(-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 degrees C, with DeltaG=34kJ/mol and DeltaH=300kJ/mol, and is, unusually, independent of protein concentration. The kinetics of thermal unfolding and refolding are slow, with activation energies of 167 and 44kJ/mol, respectively. A model for folding and assembly is discussed.     

Clitocypin, a new cysteine proteinase inhibitor, is monomeric: impact on the mechanism of folding

The molecular mass of clitocypin, a new type of cysteine proteinase inhibitor from the mushroom Clitocybe nebularis, has been determined by analytical ultracentrifugation and gel exclusion chromatography. The result is in agreement with the formula mass of 16.8 kDa, demonstrating that the inhibitor is a monomer in aqueous solution. This enables the kinetics of unfolding and refolding to be interpreted in terms of folding in a kinetically two state, highly cooperative transition from the thermally unfolded state.