AbstractWe have examined the proteolysis of bovine pancreatic ribonuclease A (RNase) by thermolysin when dissolved in aqueous buffer, pH 7.0, in the presence of 50% (v/v) trifluoroethanol (TFE). Under these solvent conditions, RNase acquires a conformational state characterized by an enhanced content of secondary structure (helix) and reduced tertiary structure, as given by CD measurements. It was found that the TFE‐resistant thermolysin, despite its broad substrate specificity, selectively cleaves the 124‐residue chain of RNase in its TFE state (20–42 °C, 6–24 h) at peptide bond Asn 34‐Leu 35, followed by a slower cleavage at peptide bond Thr 45‐Phe 46. In the absence of TFE, native RNase is resistant to proteolysis by thermolysin. Two nicked RNase species, resulting from cleavages at one or two peptide bonds and thus constituted by two (1–34 and 35–124) (RNase Th1) or three (1–34, 35–45 and 46–124) (RNase Th2) fragments linked covalently by the four disulfide bonds of the protein, were isolated to homogeneity by chromatography and characterized. CD measurements provided evidence that RNase Th1 maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (‐ΔTm 16 °C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Th1 is closely similar to that of the intact protein was confirmed unambiguously by two‐dimensional NMR measurements. Structural differences between the two protein species are located only at the level of the chain segment 30–41, i.e., at residues nearby the cleaved Asn 34‐Leu 35 peptide bond. RNase Th1 retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 31–39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41–48 region forms a β‐strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Th1 and Th2 are in line with the concept that proteins appear to tolerate extensive structural variations only at their flexible or loose parts exposed to solvent. We discuss the conformational features of RNase in its TFE‐state that likely dictate the selective proteolysis phenomenon by thermolysin.