Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains.


Abstract

The role of domains as folding units was investigated with a two-domain protein, yeast phosphoglycerate kinase. Each of the domains was produced independently by site-directed mutagenesis. It has been previously demonstrated by several criteria that these domains are able to fold in vivo into a quasi-native structure [Minard et al. (1989a) Protein Eng. 3, 55-60; Fairbrother et al. (1989) Protein Eng. 3, 5-11]. In the present study, the reversibility of the unfolding-refolding process induced by guanidine hydrochloride was investigated for the intact protein and the isolated domains. The transitions were followed by circular dichroism for both domains and the intact protein and by the variations in enzyme activity for the intact protein. Tryptophan residues were used as intrinsic conformational probes of the C-domain. An extrinsic fluorescent probe, N-[[(iodoacetyl)amino]ethyl]-8-naphthylamine-1-sulfonic acid (IAEDANS), was bound to the unique cysteinyl residue Cys97 to observe the conformational events in the N-domain. The unfolding-refolding transitions of each domain in the intact protein and in the isolated domains prepared by site-directed mutagenesis were compared. It was shown that the two domains are able to refold in a fully reversible process. A hyperfluorescent intermediate was detected during the folding of both the isolated C-domain and the intact yeast phosphoglycerate kinase. The stability of each isolated domain was found to be similar, the free energy of unfolding being approximately half that of the intact molecule. Study holds ProTherm entries: 3647, 3648, 3649 Extra Details: additive : EDTA(500 uM), domain; reversibility; transition; enzyme activity;,free energies of unfolding

Submission Details

ID: 3YfBMNcD4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:22 p.m.

Version: 1

Publication Details
Missiakas D;Betton JM;Minard P;Yon JM,Biochemistry (1990) Unfolding-refolding of the domains in yeast phosphoglycerate kinase: comparison with the isolated engineered domains. PMID:2271549
Additional Information

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 PDB Entries

Structure ID Release Date Resolution Structure Title
1FW8 2001-03-22 2.3 CIRCULARLY PERMUTED PHOSPHOGLYCERATE KINASE FROM YEAST: PGK P72
1QPG 1996-06-10 2.4 3-PHOSPHOGLYCERATE KINASE, MUTATION R65Q
3PGK 1982-09-24 2.5 The structure of yeast phosphoglycerate kinase at 0.25 nm resolution

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.5 Phosphoglycerate kinase Q6FKY1 PGK_CANGA
100.0 Phosphoglycerate kinase P00560 PGK_YEAST