Role of entropy in protein thermostability: folding kinetics of a hyperthermophilic cold shock protein at high temperatures using 19F NMR.
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.
Schuler
Benjamin
B
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany. bschuler@rz.uni-potsdam.de
Kremer
Werner
W
Kalbitzer
Hans Robert
HR
Jaenicke
Rainer
R
D001426:Bacterial Proteins / Q000737:chemistry*; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry*; D019277:Entropy; D005461:Fluorine / Q000737:chemistry*; D019791:Guanidine / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry*; D006358:Hot Temperature; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D020124:Thermotoga maritima / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; C077188:cold-shock protein CspB, Bacteria; D005461:Fluorine; D014364:Tryptophan; D019791:Guanidine