To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microorganisms, were constructed by means of site-directed mutagenesis. The far-ultraviolet (UV) CD spectra of five mutant alpha subunits with Ala in place of Pro were identical to the spectrum of the wild-type protein, the exception being the mutant at position 207 (P207A). CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (delta delta dS) at the denaturation temperature of the wild-type protein (54.1 degrees C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 13451, 13452, 13453, 13454, 13455, 13456 Extra Details: tryptophan synthase alpha subunit; conformational stability ;,site-directed mutagenesis; calorimetric measurements
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:16 p.m.
|Number of data points||67|
|Proteins||Tryptophan synthase alpha chain ; Tryptophan synthase alpha chain|
|Assays/Quantities/Protocols||Experimental Assay: dCp temp:54.1 C, details:Additives , ionic:EDTA: 1 mM ; Experimental Assay: ddG ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dCp ionic:: , details:Additives EDTA (1 mM), ; Experimental Assay: dG_H2O ; Derived Quantity: dTm ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS|