Periplasmic protein disulfide isomerase (DsbA) from Escherichia coli is a strongly oxidizing thiol reagent with one catalytic disulfide bridge and an intrinsic redox potential of -0.089 V. Gel filtration experiments and analytical ultracentrifugation studies demonstrate that DsbA is a monomeric protein with a molecular mass of 21.1 kDa, independent of its redox state. In order to investigate the molecular basis of its redox properties, the guanidinium.chloride-induced folding/unfolding equilibrium of the reduced and the oxidized form of the enzyme were compared. The transitions at pH 7.0 and 30 degrees C were found to be fully reversible and allowed the calculation of the free energy of stabilization of oxidized and reduced DsbA according to a two-state model for the unfolding transition. The analysis reveals that reduced DsbA is 22.7 (+/- 4.0) kJ/mol more stable than oxidized DsbA. This energetic difference is essentially independent of temperature, although the overall free energies of stabilization of both oxidized and reduced DsbA vary strongly between 20 and 30 degrees C as a consequence of changes in the cooperativity of the transitions The conformational tension of 22.7 (+/- 4.0) kJ/mol in oxidized DsbA quantitatively explains the oxidizing properties of the protein, as it causes a change of redox equilibrium constants between DsbA and thiols of about four orders of magnitude, corresponding to an increase of the standard redox potential of 0.118 (+/- 0.021) V. We conclude that the oxidizing properties of DsbA mainly result from a tense conformation of its oxidized form, that is converted to the relaxed, reduced state upon oxidation of thiols by DsbA. The results are discussed in terms of a general principle underlying the oxidizing properties of protein disulfide isomerases. Study holds ProTherm entries: 9268, 9269, 9270, 9271 Extra Details: additive : EDTA(50 uM), DsbA protein; protein disulfide isomerase (PDI);,protein folding; redox potential
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:37 p.m.
|Number of data points||8|
|Proteins||Thiol:disulfide interchange protein DsbA ; Thiol:disulfide interchange protein DsbA|
|Assays/Quantities/Protocols||Experimental Assay: Cm details:Additives , temp:30.0 C ; Experimental Assay: dG_H2O details:Additives , temp:30.0 C ; Experimental Assay: Cm details:Additives EDTA (50 uM),, temp:30.0 C ; Experimental Assay: dG_H2O details:Additives EDTA (50 uM),, temp:30.0 C ; Experimental Assay: Cm temp:20.0 C, details:Additives ; Experimental Assay: dG_H2O temp:20.0 C, details:Additives ; Experimental Assay: Cm details:Additives EDTA (50 uM),, temp:20.0 C ; Experimental Assay: dG_H2O details:Additives EDTA (50 uM),, temp:20.0 C|
|Libraries||Mutations for sequence AQYEDGKQYTTLEKPVAGAPQVLEFFSFFCPHCYQFEEVLHISDNVKKKLPEGVKMTKYHVNFMGGDLGKDLTQAWAVAMALGVEDKVTVPLFEGVQKTQTIRSASDIRDVFINAGIKGEEYDAAWNSFVVKSLVAQQEKAAADVQLRGVPAMFVNGKYQLNPQGMDTSNMDVFVQQYADTVKYLSEKK|