Folding,stability, and secondary structure of a new dimeric cysteine proteinase inhibitor |
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Authors: | Kidric Marjetka Fabian Heinz Brzin Joze Popovic Tatjana Pain Roger H |
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Institution: | Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia. marjetka.kidric@ijs.si |
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Abstract: | 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. |
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Keywords: | Clitocypin Cysteine proteinase inhibitors Thermal unfolding Protein stability Activation energy Dimer folding and assembly Secondary structure Circular dichroism Infrared spectroscopy |
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