Energetics of Protein Stability at Extreme Environmental Temperatures in Bacterial Trigger Factors

Abstract

Trigger factor is the first molecular chaperone interacting cotranslationally with virtually all nascent polypeptides synthesized by the ribosome in bacteria. The stability of this primary folding assistant was investigated using trigger factors from the Antarctic psychrophile Pseudoalteromonas haloplanktis, the mesophile Escherichia coli, and the hyperthermophile Thermotoga maritima. This series covers nearly all temperatures encountered by living organisms. We show that proteins adapt their stability over the whole range of biological temperatures via adjustments of the same fundamental mechanisms, involving increases in enthalpic stabilization and decreases in unfolding rates, in parallel with the environmental temperature. Enthalpic stabilization in trigger factors is characterized by large increases in the melting temperature, T(m), ranging from 33 to 96.6 °C, associated with similarly large increases in unfolding enthalpy as revealed by differential scanning calorimetry. Stopped-flow spectroscopy shows that the folding rate constants for the three investigated proteins are similar, whereas the unfolding rate constants differ by several orders of magnitude, revealing that kinetic resistance to unfolding drives adjustments of protein stability. While the unusual stability of hyperthermophilic proteins has attracted much attention, this study indicates that they are an extreme case of a more general continuum, the other extreme being represented by natively unstable proteins from psychrophiles.

Protein folding at extreme temperatures: Current issues

“…The same observation was made for a psychrophilic tryptophan synthase subunit (Fig. 2) as compared with its E. coli homologue [19] and suggests a common behavior of cold-adapted proteins. On the other hand, proteins from Adapted from [18].…”

“…Finally, the most relevant parameters, the microscopic rate constants for folding k f (H 2 O) and for unfolding k u (H 2 O) are obtained by extrapolation on the Y axis, i.e. in the absence of denaturant, in H 2 O. Reprinted from [19] by permission of Oxford University Press.…”

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

“…This substitution is consistent with the weakening of intramolecular interactions and enhanced local flexibility of cold-adapted enzymes with respect to their warm-adapted counterparts. Glycine residues are known to increase protein flexibility and are generally highly frequent in coldadapted proteins [38][39][40]48]. In contrast with this trend, glycines are overall more abundant in ApAAP (11.5%) than in SpAAP (7%) (Fig.…”

Functional adaptations of the bacterial chaperone trigger factor to extreme environmental temperatures

“…Production and purification of PhTF, EcTF and TmTF were performed essentially as described (Piette et al, 2010;Struvay et al, 2013). The genes of TFs were cloned into pET22b expression vectors (Novagen) without His-tag.…”

“…Microbial life under these extreme environmental temperatures obviously requires a vast array of adaptations at all cellular levels (Gerday and Glansdorff, 2007;Horikoshi et al, 2011). Previous studies on temperature adaptations of proteins focused mainly on enzyme activity, protein stability and folding (Vieille and Zeikus, 2001;Feller, 2010;Struvay et al, 2013). However, a key determinant of these adaptations is the acquisition of the final, biologically active conformation of proteins aided by chaperones and folding catalysts, which remains almost unexplored.…”