The cis/trans isomerization of prolyl peptide bonds has been suggested to dominate the folding of the alpha subunit of tryptophan synthase from Escherichia coli (alphaTS). To test the role of the unique cis isomer between Asp27 and Pro28, the folding properties of P28A, P28G and G(3)P28G, a three-glycine insertion mutant between Asp27 and Gly28, were investigated using urea as a denaturant. Circular dichroism analysis demonstrated that none of the mutations perturb the secondary structure significantly, although the aromatic side-chain packing is altered for P28A and P28G. All three mutant proteins inherited the three-state thermodynamic behavior observed in wild-type alphaTS, ensuring that the fundamental features of the energy surface are intact. Kinetic studies showed that neither alanine nor glycine substitutions at Pro28 results in the elimination of any slow-refolding phases. By contrast, the G(3)P28G mutant eliminates the fastest of the slow-refolding phases and one of the two unfolding phases. Double-jump experiments on G(3)P28G confirm the assignment of the missing refolding phase to the isomerization of the Asp27-Pro28 peptide bond. These results imply that the local stability conveyed by the tight, overlapping turns containing the cis peptide bond is sufficient to favor the cis isomer for several non-prolyl residues. The free energy required to drive the isomerization reaction is provided by the formation of the stable intermediate, demonstrating that the acquisition of structure and stability is required to induce subsequent rate-limiting steps in the folding of alphaTS. Study holds ProTherm entries: 17622, 17623, 17624, 17625, 17626, 17627 Extra Details: alpha subunit, 0.2 mM K2EDTA and 1mM betaME were added in the experiment, N->I. kinetic folding mechanism; mutagenesis; parallel folding channels
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:49 p.m.
|Number of data points||22|
|Proteins||Tryptophan synthase alpha chain ; Tryptophan synthase alpha chain|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O|
|Libraries||Mutations for sequence MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS|