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.

Experimental determination of upper bound for transition path times in protein folding from single-molecule photon-by-photon trajectories

“…The mean FRET efficiency for the immobilized and freely diffusing molecules is virtually identical, showing that the root mean-squared distance between the dyes (Table S1) in the unfolded state is the same. This distance increases with increasing denaturant concentration as observed for several other proteins (19,(25)(26)(27)(28)(29).…”

Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations

“…Here we demonstrate the quantitative applicability of single-molecule FRET measurements to unfolded proteins using intensity and life-time measurements in combination with molecular simulations for two well characterized two-state proteins, the 64-residue ␣/␤ protein L (Fig. 1) and the 66-residue all-␤ cold-shock protein CspTm (18)(19)(20)(21)(22)(23). The size distribution of the unfolded state was determined for both proteins, and for protein L it was compared with equilibrium and time-resolved small-angle x-ray scattering (SAXS) experiments (1,20).…”

Different packing of external residues can explain differences in the thermostability of proteins from thermophilic and mesophilic organisms

“…The study of protein stability can help explore the sequencestructure stability relationship (Kumar et al, 2001;Olofsson et al, 2007;Perl et al, 1998;Razvi and Scholtz, 2006;Schuler et al, 2002). It has been shown that no correlations exist between a protein's melting temperature and parameters, such as change in heat capacity, change in accessible surface area upon folding and number of residues in the protein (Kumar et al, 2001).…”

“…Moreover, most proteins use different combinations of these three general approaches, and so a simple classification of proteins into three separate groups was not possible (Razvi and Scholtz, 2006). It has been shown that the folding rates of cold shock proteins from Thermotoga maritima (thermophilic organism) and Bacillus subtilis (mesophilic organism) are similar, while the unfolding rate of the thermophilic protein is two orders lower than that of its mesophilic homologue (Perl et al, 1998;Schuler et al, 2002). Thus, distinctions in stability arise from distinctions in the unfolding rate constants of thermophilic and mesophilic proteins.…”