Conformational Stability of Ribonuclease T1I. Thermal Denaturation and Effects of Salts1

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Abstract
The thermal transition of RNase T1 was studied by two different methods; tryptophan residue fluorescence and circular dichroism. The fluorescence measurements provide information about the environment of the indole group and CD measurements on the gross conformation of the polypeptide chain. Both measurements at pH 5 gave the same transition temperature of 56°C and the same thermodynamic quantities, ΔHtr (=120kcal/mol) and ΔStr (=360 eu/mol), for the transition from the native state to the thermally denatured state, indicating simultaneous melting of the whole molecule including the hydrophobic region where the tryptophan residue is buried. Stabilization by salts was observed in the pH range from 2 to 10, since the presence of 0.5 m NaCl caused an increase of about 5°C to 10°C in the transition temperature, depending on the pH. The fluorescence measurements on the RNase T1 complexed with 2'-GMP showed a transition with ΔHtr=167 kcal/mol and ΔStr=497 eu/mol at a transition temperature about 6°C higher than that for the free enzyme. The large value of ΔHtr for RNase T1 indicates the highly cooperative nature of the thermal transition; this value is much higher than those of other globular proteins. Analysis of the CD spectrum of thermally denatured RNase T1 suggests that the denatured state is not completely random but retains some ordered structures.

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