Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit.

Abstract

To elucidate the role of conserved proline residues of the tryptophan synthase alpha subunit from Escherichia coli in stability and folding, equilibrium and kinetic studies of the unfolding-refolding induced by guanidine hydrochloride for six mutant alpha subunits (Pro-->Ala) were carried out by peptidyl circular dichroism and aromatic fluorescence measurements at pH 7 and 25 degrees C. These results were analyzed assuming the presence of one intermediate (I) state in the denaturation process. (I) For all mutant and wild-type proteins, the Gibbs energy change (delta Gni(H2O)) in water between the native (N) and I states coincided with the difference (delta G++u(H2O)-delta G++r(H2O)) between the activation Gibbs energy changes in water for the unfolding (delta G++u(H2O) and refolding (delta G++r(H2O) reactions. This means that the early folding intermediate of the alpha subunit corresponds to the equilibrium intermediate. Delta Gni(H2O) values of all mutant proteins decreased compared with that of the wild-type protein. Gibbs energy change (delta Gid(H2O) in water between I and the denatured (D) states was not substantially affected by the substitutions. Delta G++u(H2O) and delta G++r(H2O) decreased and increased, respectively, for all mutant proteins. (2) Six conserved prolines played roles in stability and folding of the alpha subunit in a different manner: prolines 28 and 96 by stabilizing the N state and prolines 28, 96, 132, and 207 by destabilizing the I state. The contributions of prolines 57 and 62 to the stability were marginal. (3) Cis proline 28 was not the origin of the slow phase in the refolding kinetics assumed to arise from the cis-trans isomerization reaction of proline. Study holds ProTherm entries: 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 2285, 2286, 2287, 2288, 2289, 2290, 2291 Extra Details: additive : EDTA(0.1 mM),N -> I tryptophan synthase alpha subunit; Gibbs energy changes;,kinetic studies; circular dichroism

Submission Details

ID: 5Ww7KTD33

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:16 p.m.

Version: 1

Publication Details
Ogasahara K;Yutani K,Biochemistry (1997) Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit. PMID:9020793
Additional Information

Study Summary

Number of data points 39
Proteins Tryptophan synthase alpha chain ; Tryptophan synthase alpha chain
Unique complexes 7
Assays/Quantities/Protocols Experimental Assay: dG_H2O ; Derived Quantity: ddG_H2O
Libraries Mutations for sequence MERYESLFAQLKERKEGAFVPFVTLGDPGIEQSLKIIDTLIEAGADALELGIPFSDPLADGPTIQNATLRAFAAGVTPAQCFEMLALIRQKHPTIPIGLLMYANLVFNKGIDEFYAQCEKVGVDSVLVADVPVEESAPFRQAALRHNVAPIFICPPNADDDLLRQIASYGRGYTYLLSRAGVTGAENRAALPLNHLVAKLKEYNAAPPLQGFGISAPDQVKAAIDAGAAGAISGSAIVKIIEQHINEPEKMLAALKVFVQPMKAATRS

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Tryptophan synthase alpha chain C4ZTV3 TRPA_ECOBW
100.0 Tryptophan synthase alpha chain B1XBK9 TRPA_ECODH
100.0 Tryptophan synthase alpha chain A7ZZJ6 TRPA_ECOHS
100.0 Tryptophan synthase alpha chain B1ITJ5 TRPA_ECOLC
100.0 Tryptophan synthase alpha chain P0A877 TRPA_ECOLI
100.0 Tryptophan synthase alpha chain P0A878 TRPA_SHIFL
99.6 Tryptophan synthase alpha chain B7LY16 TRPA_ECO8A
99.6 Tryptophan synthase alpha chain B6I9X4 TRPA_ECOSE
99.6 Tryptophan synthase alpha chain B7L492 TRPA_ECO55
99.3 Tryptophan synthase alpha chain A7ZL78 TRPA_ECO24
99.6 Tryptophan synthase alpha chain B2U0F1 TRPA_SHIB3
99.3 Tryptophan synthase alpha chain Q8FHW0 TRPA_ECOL6
99.6 Tryptophan synthase alpha chain Q31ZV3 TRPA_SHIBS
99.6 Tryptophan synthase alpha chain Q0T5D6 TRPA_SHIF8
98.9 Tryptophan synthase alpha chain Q3Z108 TRPA_SHISS
99.3 Tryptophan synthase alpha chain Q32GT0 TRPA_SHIDS
98.9 Tryptophan synthase alpha chain Q8X7B5 TRPA_ECO57
98.9 Tryptophan synthase alpha chain B5YZP0 TRPA_ECO5E
98.5 Tryptophan synthase alpha chain B7ML76 TRPA_ECO45
98.5 Tryptophan synthase alpha chain A1AAN0 TRPA_ECOK1
98.5 Tryptophan synthase alpha chain Q1RCA7 TRPA_ECOUT
98.9 Tryptophan synthase alpha chain B7UR66 TRPA_ECO27
98.1 Tryptophan synthase alpha chain B7MU99 TRPA_ECO81
98.5 Tryptophan synthase alpha chain Q0TIB0 TRPA_ECOL5
98.5 Tryptophan synthase alpha chain B7N473 TRPA_ECOLU
98.1 Tryptophan synthase alpha chain B1LH32 TRPA_ECOSM
97.8 Tryptophan synthase alpha chain B7NVN0 TRPA_ECO7I
97.0 Tryptophan synthase alpha chain B7LS20 TRPA_ESCF3