Guanidine hydrochloride (GdnHCl) and thermally induced unfolding measurements on the oxidized form of Escherichia coli thioredoxin at pH 7 were combined for the purpose of assessing the functional dependence of unfolding free energy changes on denaturant concentration over an extended GdnHCl concentration range. Conventional analysis of GdnHCl unfolding exhibits a linear plot of unfolding delta G vs [GdnHCl] in the transition zone. In order to extend unfolding delta G measurements outside of that narrow concentration range, thermal unfolding measurements were performed using differential scanning calorimetry (DSC) in the presence of low to moderate concentrations of GdnHCl. The unfolding delta G values from the DSC measurements were corrected to 25 degrees C using the Gibbs-Helmholtz equation and mapped onto the delta G vs [GdnHCl] plot. The dependence of unfolding delta G on [GdnHCl] was found to be linear over the full denaturant concentration range, provided that the chloride ion concentration was kept at a threshold of greater than or equal to 1.5 M. In the DSC experiments performed in the presence of GdnHCl, chloride concentrations were maintained at 1.5 M by addition of appropriate amounts of NaCl. The linear extrapolation method (LEM) gives an unfolding free energy change in the absence of denaturant (delta G degrees N-U) in excellent agreement with the delta G determined by DSC measurement in 1.5 M NaCl. The various methods give a consensus unfolding delta G value of 8.0 kcal/mol at 25 degrees C in the absence of denaturant.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4186, 4187, 4188 Extra Details: functional dependence; transition zone;,Gibbs-Helmholtz equation; N-U equilibrium
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:24 p.m.
|Number of data points||8|
|Proteins||Thioredoxin 1 ; Thioredoxin 1|
|Assays/Quantities/Protocols||Experimental Assay: Cm ionic:NaCl: 0.5 M ; Experimental Assay: m ionic:NaCl: 0.5 M ; Experimental Assay: dG_H2O ionic:NaCl: 0.5 M ; Experimental Assay: Cm ionic:: ; Experimental Assay: m ionic:: ; Experimental Assay: dG_H2O ionic:: ; Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA|