Stability mutants of staphylococcal nuclease: Large compensating enthalpy-entropy changes for the reversible denaturation reaction

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Abstract

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change ΔHapp on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 ± 0.2 °C and a ΔHapp of 86.8 ± 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 °C and 96 ± 2 kcal/mol. The heat capacity change on denaturation ΔCp, was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of ΔHapp and ΔSapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated ΔCp for the mutant proteins and the d(ΔGapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.

Original languageEnglish (US)
Pages (from-to)4761-4768
Number of pages8
JournalBiochemistry®
Volume27
Issue number13
StatePublished - 1988

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Micrococcal Nuclease
Denaturation
Entropy
Enthalpy
Guanidine
Temperature
Hot Temperature
Mutant Proteins
Amino Acid Substitution
Urea
Fluorescence
Mutation
Solvation
Equilibrium constants
Free energy
Specific heat
Substitution reactions
Amino Acids

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Stability mutants of staphylococcal nuclease: Large compensating enthalpy-entropy changes for the reversible denaturation reaction",
abstract = "By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change ΔHapp on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 ± 0.2 °C and a ΔHapp of 86.8 ± 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 °C and 96 ± 2 kcal/mol. The heat capacity change on denaturation ΔCp, was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of ΔHapp and ΔSapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated ΔCp for the mutant proteins and the d(ΔGapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.",
author = "Shortle, {David R} and Meeker, {Alan Keith} and Freire, {Ernesto I}",
year = "1988",
language = "English (US)",
volume = "27",
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journal = "Biochemistry",
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T2 - Large compensating enthalpy-entropy changes for the reversible denaturation reaction

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PY - 1988

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AB - By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change ΔHapp on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 ± 0.2 °C and a ΔHapp of 86.8 ± 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 °C and 96 ± 2 kcal/mol. The heat capacity change on denaturation ΔCp, was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of ΔHapp and ΔSapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated ΔCp for the mutant proteins and the d(ΔGapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.

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