In contrast to the folding reaction of proteins, unfolding is a highly cooperative process and intermediates are hard to detect. Nevertheless, in reversal of folding, unfolding is a stepwise process as well and starts at confined, so-called first unfolding regions'. Consequently, stabilization of these unfolding regions should result in a significant deceleration of the unfolding process, which bears significance for the technical application of enzymes as unfolding reactions often are followed by irreversible steps. Limited proteolysis proved to be a sensitive tool for analyzing both local and global conformational changes of proteins. By analysis of complementary N- and C-terminal fragments, which have been obtained by proteolysis under unfolding conditions, the first unfolding region(s) can be identified. Here, the region between the first α-helix and the first β-strand of bovine pancreatic ribonuclease A (RNase A) could be assigned as unfolding region. Moreover, limited proteolysis can be used to determine the rate constant of the unfolding reaction ku under conditions where unfolding is slow compared to proteolysis. The main advantage over conventional spectroscopic methods is the direct determination of ku even under native-like conditions. By comparing the kinetic stability with the thermodynamic stability, the effect of modifications on the native or unfolded state can be dissected. Or, in other words, the involvement of the regions concerned into the various stages of folding and unfolding. Modifications of the unfolding region of RNase A in fact revealed a pronounced impact on the native state and the unfolding reaction. Non-natural structural mimics (prostheses), which have been incorporated by expressed protein ligation to stabilize the RNase A molecule by performing the folding region, could indeed be shown to markedly affect the unfolded state and the folding reaction.