Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using 19F NMR.


Abstract

We used (19)F NMR to extend the temperature range accessible to detailed kinetic and equilibrium studies of a hyperthermophilic protein. Employing an optimized incorporation strategy, the small cold shock protein from the bacterium Thermotoga maritima (TmCsp) was labeled with 5-fluorotryptophan. Although chaotropically induced unfolding transitions revealed a significant decrease in the stabilization free energy upon fluorine labeling, the protein's kinetic folding mechanism is conserved. Temperature- and guanidinium chloride-dependent equilibrium unfolding transitions monitored by (19)F NMR agree well with the results from optical spectroscopy, and provide a stringent test of the two-state folding character of TmCsp. Folding and unfolding rate constants at high temperatures were determined from the (19)F NMR spectra close to the midpoint of thermal unfolding by global line shape analysis. In combination with results from stopped-flow experiments at lower temperatures, they show that the folding rate constant of TmCsp and its temperature dependence closely resemble those of its mesophilic homologue from Bacillus subtilis, BsCspB. However, the unfolding rate constant of TmCsp is two orders of magnitude lower over the entire temperature range that was investigated. Consequently, the difference in conformational stability between the two proteins is solely due to the unfolding rate constant over a wide temperature range. A thermodynamic analysis points to an important role of entropic factors in the stabilization of TmCsp relative to its mesophilic homologues. Study holds ProTherm entries: 15642 Extra Details: 0.2 mM EDTA, 1 mM DTE were added in the experiment. Hyperthermophilic; free energy; folding rate constant; entropic factors

Submission Details

ID: d2q4C6Ga3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Schuler B;Kremer W;Kalbitzer HR;Jaenicke R,Biochemistry (2002) Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using 19F NMR. PMID:12269809
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
1G6P 2001-11-07 SOLUTION NMR STRUCTURE OF THE COLD SHOCK PROTEIN FROM THE HYPERTHERMOPHILIC BACTERIUM THERMOTOGA MARITIMA

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cold shock-like protein O54310 CSP_THEMA