STABILITY OF RECOMBINANT LYS25-RIBONUCLEASE-T1

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn·HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the ΔG° values derived from the three different experimental techniques at pH 5, ϑ = 25 °C: DSC, 46.6 ± 2.1 kJ/mol; UV melting curves, 48.7 ± 5 kJ/mol; Gdn·HCl transition curves, 40.8 ± 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van’t Hoff and calorimetric enthalpy, ΔHvH/ΔHcal, is 0.97 ± 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by ΔCp° = 5.1 ± 0.5 kJ/(mol·K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction. The isoenzyme Gln25-ribonuclease T1 shows lower stability under comparable conditions (Shirley et al., 1989). Energy minimization calculations provide a rationalization for this finding by suggesting the existence of a salt bridge between Glu28 and Lys25 in Lys25-ribonuclease T1 that cannot form in the Gln25-ribonuclease T1 isoenzyme. © 1990, American Chemical Society. All rights reserved.