Limited proteolysis of ribonuclease A with thermolysin in trifluoroethanol

Abstract

We 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 Thl) 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 Thl maintains the overall conformational features of the native protein, but shows a reduced thermal stability with respect to that of the intact species (-ATm 16 "C); RNase Th2 instead is fully unfolded at room temperature. That the structure of RNase Thl 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 Thl retained about 20% of the catalytic activity of the native enzyme, whereas RNase Th2 was inactive. The 3 1-39 segment of the polypeptide chain in native RNase forms an exposed and highly flexible loop, whereas the 41-48 region forms a &strand secondary structure containing active site residues. Thus, the conformational, stability, and functional properties of nicked RNase Thl 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.Keywords: CD; limited proteolysis; NMR; ribonuclease A; thermolysin; trifluoroethanol Short-chain alcohols, such as methanol, ethanol, propanol, chloroethanol, and, in particular, trifluoroethanol, have been used frequently to induce a-helical conformations in otherwise randomly coiled or partially structured polypeptides (Toniolo et al., 1975;

Comparative studies on trifluoroethanol (TFE) state of a thermophilic α-amylase and its mesophilic counterpart: limited proteolysis, conformational analysis, aggregation and reactivation of the enzymes

“…Presumably, in the absence of TFE, the 29 kDa fragment undergoes further proteolysis to yield the 17 and 13 kDa fragments. The resistance of thermolysin, a thermophilic protease, in the presence of TFE and organic solvents has been previously noted [23,34,35] and our results showed that thermolysin retains 90% of its initial activity in 40% of TFE concentration.…”

“…The results of this study and previous investigations [23,34] show that the proteolysis of proteins by thermolysin in the presence of TFE can be used to achieve selective fragmentation of proteins. As shown by Fontana et al [23,34], limited digestion of proteins such as cytochrome c, lysozyme and ribonuclease A with thermolysin in the presence of TFE can be considered as a valuable procedure to produce large fragments which can be used for sequencing [35] and evaluation of structure-activity relationship in proteins [32,33].…”

“…1). The digestion pattern of BAA shows seven clear bands (42,34,29,24,17,15 and 13 kDa) whilst only three bands have been seen in BLA. In the presence of TFE, BAA and BLA are digested into relatively few protein fragments.…”

“…Apparently both ␣-amylases dissolved in 40% TFE, are preferentially cleaved by thermolysin at a single peptide bond, whereas no difference in the digestion pattern of these two enzymes has been seen. In addition, in the presence of 10% TFE, digestion of BAA is limited and five fragments (42,34,29,24 and 15 kDa) can be seen in its digestion pattern. It is likely that the faint 29 kDa band at 0% TFE becomes more prominent at 10% TFE, while the reverse occurs with respect to the 17 and 13kDa fragments.…”

“…As shown by Fontana et al [23,34], limited digestion of proteins such as cytochrome c, lysozyme and ribonuclease A with thermolysin in the presence of TFE can be considered as a valuable procedure to produce large fragments which can be used for sequencing [35] and evaluation of structure-activity relationship in proteins [32,33].…”