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Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit.
Biochemistry. 1997 Jan 28; 36(4):932-40.B

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.

Authors+Show Affiliations

Ogasahara K
Institute for Protein Research, Osaka University, Japan.
Yutani K
No affiliation info available

MeSH

Conserved SequenceEnzyme StabilityEscherichia coliGuanidineGuanidinesKineticsModels, MolecularMolecular StructureMutagenesis, Site-DirectedPoint MutationProlineProtein ConformationProtein DenaturationProtein FoldingThermodynamicsTryptophan Synthase

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9020793

Citation

Ogasahara, K, and K Yutani. "Equilibrium and Kinetic Analyses of Unfolding and Refolding for the Conserved Proline Mutants of Tryptophan Synthase Alpha Subunit." Biochemistry, vol. 36, no. 4, 1997, pp. 932-40.
Ogasahara K, Yutani K. Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit. Biochemistry. 1997;36(4):932-40.
Ogasahara, K., & Yutani, K. (1997). Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit. Biochemistry, 36(4), 932-40.
Ogasahara K, Yutani K. Equilibrium and Kinetic Analyses of Unfolding and Refolding for the Conserved Proline Mutants of Tryptophan Synthase Alpha Subunit. Biochemistry. 1997 Jan 28;36(4):932-40. PubMed PMID: 9020793.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Equilibrium and kinetic analyses of unfolding and refolding for the conserved proline mutants of tryptophan synthase alpha subunit. AU - Ogasahara,K, AU - Yutani,K, PY - 1997/1/28/pubmed PY - 1997/1/28/medline PY - 1997/1/28/entrez SP - 932 EP - 40 JF - Biochemistry JO - Biochemistry VL - 36 IS - 4 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9020793/Equilibrium_and_kinetic_analyses_of_unfolding_and_refolding_for_the_conserved_proline_mutants_of_tryptophan_synthase_alpha_subunit_ L2 - https://doi.org/10.1021/bi961660c DB - PRIME DP - Unbound Medicine ER -