In the first part of this work, the stability and folding of RNase A and its proteolytically more resistant variant A20P-RNase A was investigated in trifluoroethanol (TFE). In addition to the characterization of the proteins by spectroscopy the method of limited proteolysis was used to detect local structural changes and to determine rate constants of unfolding. In TFE concentrations up to 20 % (v/v), a decrease in flexibility of the loop-region around Ala20 was observed by limited proteolysis. This finding was attributed to a stabilization of the helices surrounding the loop-region by TFE. In addition to the characterization of the thermodynamic equilibrium unfolding and refolding reactions of RNase A and A20P-RNase A in TFE were investigated by stopped flow spectroscopy. These investigations were completed by limited proteolysis to determine unfolding rate constants in 0 - 45 % (v/v) TFE. The determination of the rate constants of unfolding under native and native-like conditions led to the identification of a native-like state. In the second part of this work, the model of the unfolding region developed in our group should be verified by mutational studies. For this purpose, the influence of mutations in two proteolytically sensitive regions of RNase A on the thermodynamic stability of the enzyme was investigated: the loop region around Ala20, which is attacked by the unspecific proteases subtilisin Carlsberg, proteinase K, and elastase even under native conditions and the region from Lys31 to Phe46, that was identified as the so-called unfolding region and becomes as first proteolytically accessible under denaturing conditions. While mutations in the region around Ala20 hardly influenced the thermodynamic stability of the RNase A, the mutations in the postulated unfolding region led to a drastic destabilization of the entire molecule. The results of our investigations show that the region Lys31-Phe46 contributes considerably to the stability of the entire protein molecule and support the model of the unfolding region. |