Stability of recombinant Lys25-ribonuclease T1

Abstract

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 delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees 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, delta HvH/delta Hcal, 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 delta Cp degrees = 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.(ABSTRACT TRUNCATED AT 250 WORDS)

X‐ray crystallographic and calorimetric studies of the effects of the mutation Trp59→ Tyr in ribonuclease T1

“…The thermodynamic quantities for the wild-type protein are in fair agreement with data published for the Lys2S isoform (Kiefhaber et al, 1990) when taking into account the different absorption coefficients used in concentration determination. Furthermore, the data confirm results obtained for the Gln2.5 form (Hu et al, 1992) when regarding the stability difference on the Lys25Gln mutation (Pace et a]., 1989) and the different pH characteristics of the two forms.…”

Thermal stabilization of ribonuclease T1 by carboxymethylation at Glu‐58 as revealed by 1H nuclear magnetic resonance spectroscopy

“…The enthalpy changes obtained by plotting as shown in the inset of Fig. 4 were about 530 + 40 kJ/mol for intact RNase Tl and about 780 + 50 kJ/mol for CM-RNase T,, The value estimated for intact RNase T, was consistent with that (490-520 kJ/mol at pH 5) obtained by other methods [4,8,10,11]. Thus, the carboxymethylation also makes the thermal unfolding more cooperative.…”

“…In CM-RNase Tt, the formation of an additional salt bridge between CM-Glu-58 and Arg-77, hence between the two domains, is thought to stabilize the whole molecule. This explanation is in line with the fact that the conformation of RNase T~ is extremely stabilized by salts [4][5][6]8,10]. In addition, a local conformational change induced by the carboxymethylation may also contribute somewhat to the stabilization.…”

“…4), suggest that practically all parts of the main and side chains were unfolded simultaneously for both intact and CM-RNase Tl. The midpoint temperature of intact RNase TI was a little lower than those obtained by calorimetry, fluorescence and CD measurements [4,8,10,11]. NMR spectroscopy detects local conformational changes at the atomic level, while other methods provide global information of the molecule.…”

“…It is a single-chain globular protein of 104 amino acid residues and is unfolded reversibly by heating or addition of denaturants. Therefore, a number of studies on the stability and unfolding/refolding have been made by using differential scanning calorimetry and spectroscopic methods including circular dichroism and fluorescence measurements [4][5][6][7][8][9][10][11]. The thermodynamic analysis indicated that the unfolding equilibrium of RNase T~ is described by a two-state model [4,5,8].…”