The concept of the unfolding region for approaching the mechanisms of enzyme stabilization

Starting from the hypothesis that chemical or genetic modifications as meth ods of protein stabilization are most successful if the most labile region of the molecule is strengthened, the importance of local structural regions and their modification for thermal stability has been investigated on two enzyme systems. The thermal unfolding of ribonuclease A and its glycosylate d form, ribonuclease B, as well as of partially deglycosylated derivatives of ribonuclease B was studied by limited proteolysis. From the analysis of the primary proteolytic cleavage sites and the kinetic stabilities, it can be concluded that the unfolding pathway of ribonuclease A is not changed by the carbohydrate attachment and that the higher stability of ribonuclease B results from the first N-acetylglucosamine residue. On neutral protease f rom Bacillus stearothermophilus where a surface-located region (residues 56 to 69) is known to be crucial for the thermal stability of the enzyme, the introduction of a disulfide bridge into this region by site-directed mutag enesis resulted in an extreme stabilization of the molecule. Cys residues, which were introduced into different positions of the protein surface, enab led the specific immobilization of the corresponding single mutant enzymes via their SH groups to Activated Thiol-Sepharose. The resulting stabilizati on effects were highest if the attachment of the enzyme to the carrier was within the residues 56 to 69. (C) 1999 Elsevier Science B.V. All rights res erved.