The urea-induced unfolding of the inactive single mutants Tyr-175----Cys and Gly-211----Glu and the active double mutant Cys-175/Glu-211 of the alpha subunit of tryptophan synthase from Escherichia coli was examined by using ultraviolet difference spectroscopy. Equilibrium techniques were used to determine the equilibrium free energies of unfolding for the mutant proteins to permit comparison with the wild-type protein. The sum of the changes in stability for the single mutants is not equal to the change seen in the double mutant. This inequality is evidence for a structural interaction between these two residues. Kinetic studies show that this synergism, which destabilizes the native form by 1.5-2.0 kcal/mol at pH 7.8, 25 degrees C, occurs only after the final rate-limiting step of domain association. Study holds ProTherm entries: 3850, 3851, 3852, 3853, 3854, 3855, 3856, 3857 Extra Details: additive : EDTA(0.2 mM),transition is from native to intermediate equilibrium techniques; structural interaction;,rate-limiting step; domain association
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:23 p.m.
|Number of data points||22|
|Proteins||Tryptophan synthase alpha chain ; Tryptophan synthase alpha chain|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: Cm ; Experimental Assay: m|
|Libraries||Mutations for sequence MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS|