Role of Entropy in Protein Thermostability: Folding Kinetics of a Hyperthermophilic Cold Shock Protein at High Temperatures Using 19F NMR

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• 作者：Benjamin Schuler ; Werner Kremer ; Hans Robert Kalbitzer ; and Rainer Jaenicke
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：October 1, 2002
• 年：2002
• 卷：41
• 期：39
• 页码：11670 - 11680
• 全文大小：264K
• 年卷期：v.41,no.39(October 1, 2002)
• ISSN：1520-4995

文摘

We used ¹⁹F NMR to extend the temperature range accessible to detailed kinetic and equilibriumstudies of a hyperthermophilic protein. Employing an optimized incorporation strategy, the small coldshock protein from the bacterium Thermotoga maritima (TmCsp) was labeled with 5-fluorotryptophan.Although chaotropically induced unfolding transitions revealed a significant decrease in the stabilizationfree energy upon fluorine labeling, the protein's kinetic folding mechanism is conserved. Temperature-and guanidinium chloride-dependent equilibrium unfolding transitions monitored by ¹⁹F NMR agree wellwith the results from optical spectroscopy, and provide a stringent test of the two-state folding characterof TmCsp. Folding and unfolding rate constants at high temperatures were determined from the ¹⁹F NMRspectra close to the midpoint of thermal unfolding by global line shape analysis. In combination withresults from stopped-flow experiments at lower temperatures, they show that the folding rate constant ofTmCsp and its temperature dependence closely resemble those of its mesophilic homologue from Bacillussubtilis, BsCspB. However, the unfolding rate constant of TmCsp is two orders of magnitude lower overthe entire temperature range that was investigated. Consequently, the difference in conformational stabilitybetween the two proteins is solely due to the unfolding rate constant over a wide temperature range. Athermodynamic analysis points to an important role of entropic factors in the stabilization of TmCsprelative to its mesophilic homologues.