TY - JOUR
T1 - Temperature and Guanidine Hydrochloride Dependence of the Structural Stability of Ribonuclease T1
AU - Plaza del Pino, Isabel M.
AU - Pace, C. Nick
AU - Freire, Ernesto
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1992/2/1
Y1 - 1992/2/1
N2 - The thermal unfolding of ribonuclease T1has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 °;C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 °C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.
AB - The thermal unfolding of ribonuclease T1has been studied by high-sensitivity differential scanning calorimetry as a function of temperature, [GuHCl], and scanning rate. The destabilizing effect of GuHCl has revealed that the kinetics of the unfolding transition become extremely slow as the transition temperature decreases. At pH 5.3 and zero GuHCl, the unfolding transition is centered at 59.1 °;C; upon increasing the GuHCl concentration, the transition occurs at lower temperatures and exhibits progressively slower kinetics; so, for example, at 3 M GuHCl, the transition temperature is 40.6 °C and is characterized by a time constant close to 10 min. Under all conditions studied (pH 5.3, pH 7.0, [GuHCl] < 3 M), the transition is thermodynamically reversible. The slow kinetics of the transition induce significant distortions in the shape of the transition profiles that can be mistakenly interpreted as deviations from a two-state mechanism. Determination of the thermodynamic parameters from the calorimetric data has required the development of an analytical formalism that explicitly includes the thermodynamics as well as the kinetics of the transition. Using this formalism, it is shown that a two-state slow-kinetics model is capable of accurately describing the structural stability of ribonuclease T1as a function of temperature, GuHCl concentration, and scanning rate. Multidimensional analysis of the calorimetric data has been used to estimate the intrinsic thermodynamic parameters for protein stability, the interaction parameters with GuHCl, and the time constant for the unfolding transition and its temperature dependence.
UR - http://www.scopus.com/inward/record.url?scp=0026454646&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026454646&partnerID=8YFLogxK
U2 - 10.1021/bi00160a033
DO - 10.1021/bi00160a033
M3 - Article
C2 - 1445858
AN - SCOPUS:0026454646
SN - 0006-2960
VL - 31
SP - 11196
EP - 11202
JO - Biochemistry
JF - Biochemistry
IS - 45
ER -