Tuning the heterogeneous early folding dynamics of phosphoglycerate kinase.


Abstract

We recently reported stretched kinetics during the formation of a collapsed, long-lived intermediate state of the large two-domain enzyme phosphoglycerate kinase (PGK). It was postulated that intrinsic roughness of the energy landscape on the way downhill to the intermediate causes the lack of a single time-scale. Here, we investigate several alternative explanations for stretched refolding dynamics in more detail: tyrosine fluorescence, multiple tryptophan probes, and rate differences between independently folding domains. To this end, we systematically simplify PGK in several steps from the full protein with two tryptophan residues and all tyrosine residues probed, to a single domain with only one tryptophan residue and no tyrosine residue probed. The kinetics in the 10 micros to 10 ms range are revealed by laser-induced temperature-jump relaxation experiments. The isolated N-terminal domain forms an intermediate by nearly single-exponential kinetics, but the isolated C-terminal domain shows strongly non-exponential kinetics. Thus, domain interaction and a cis-proline residue between the two domains are ruled out as the sole contributors to heterogeneity during the earliest folding dynamics of the C-terminal domain. We apply two limiting models for the roughness of the energy landscape. A sequential three-state model lumps all the roughness into a single trap. The "strange kinetics" model with logarithmic oscillations developed by Klafter and co-workers distributes the roughness over a larger number of states. Both models explain our data about equally well, but the coincidental values of rate constants in all of our double-exponential fits, and the absence of a spectroscopic signature distinct from the endpoints of the folding process favors more roughness than can be explained by just a single trap. Study holds ProTherm entries: 16774, 16775, 16776, 16777, 16778, 16779, 16780, 16781 Extra Details: 1 mM EDTA and 1 mM DTT were added in the experiment. Histidine-tagged variant of yeast phosphoglycerate kinase. phosphoglycerate kinase; temperature-jump; downhill folding; logarithmic oscillation; energy landscape

Submission Details

ID: Zikop5ai

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:48 p.m.

Version: 1

Publication Details
Osváth S;Sabelko JJ;Gruebele M,J. Mol. Biol. (2003) Tuning the heterogeneous early folding dynamics of phosphoglycerate kinase. PMID:14516752
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