Early studies suggested that the Escherichia coli tryptophan synthase alpha-subunit unfolded in a two-step process in which there was a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268). More recent evidence has indicated that such a structure for the intermediate seems unlikely. In this report, single Trp residues (absent in the wild-type alpha-subunit) are substituted separately for Phe residues at positions 139 (in alpha-1) and 258 (in alpha-2) to produce the F139W, F258W, and F139W/F258W mutant alpha-subunits. The UV absorbance and fluorescence properties of the F139W/F258W double mutant are identical with those of equimolar mixtures of the single mutants, suggesting that the Trp residue at each position can independently report the behavior of its respective folding unit. Each mutant alpha-subunit is wild-type enzymatically, and when UV absorbance is monitored, the urea-induced unfolding of the three tryptophan-containing alpha-subunits is virtually identical to the wild-type protein. These wild-type properties make these proteins attractive candidates for a fluorescence examination of the behavior of the individual folding units and the structure of potential intermediate(s) and as host proteins for the insertion of our existing destabilizing and/or stabilizing mutational alterations. Study holds ProTherm entries: 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199 Extra Details: additive : Na2EDTA(0.2 mM),N -> I two-step process; stable intermediate; Trp residue;,individual folding units
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:29 p.m.
|Number of data points||36|
|Proteins||Tryptophan synthase alpha chain ; Tryptophan synthase alpha chain|
|Assays/Quantities/Protocols||Experimental Assay: ddG ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS|