# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 3 Discussion  
### Thermophilic Multidomain IF2 Undergoes Cold Denaturation  
Therophilic proteins are usually heat-stable proteins and, hence, attractive for industrial applications. Is a high melting temperature always a good indicator of stability at room temperature? Here, we report that this is not always the case due to the cold-denatured state of proteins, which is little-understood compared with the heat-denatured state. A cold-denatured state may also have some physiological relevance, as indicated by the recent findings of [PERSON] et al. [36]. Interestingly, the ordering of water molecules around the unfolded protein substrates in the GroEL/ES chaperonin cage resembles a cold-denatured state [36]. Compared with thermal heat denaturation studies, cold denaturation studies are more sparse because they can only be observed under specific experimental conditions. By employing large reversibly denatured proteins from thermophilic bacteria, we found conditions where the protein undergoes cold denaturation. It should be noted that in the absence of destabilizing agents, no cold denaturation was observed. However, for industrial applications, proteins and enzymes are often used in the presence of organic solvents, at nonphysiological pHs, and ionic liquids. These can strongly destabilize the protein and, hence, may resemble the conditions used in our experiments. In our case, cold denaturation of IF2 was observed in the presence of high urea concentrations and also in guanidinium chloride. High urea concentrations were initially used to suppress aggregation of the C domain. We observed in one of our experiments that the protein was largely unfolded at ambient temperatures and refolded at higher temperatures. This effect was later confirmed for IF2 and for the G fragment. Urea enabled us to examine the molecular properties of cold denaturation, which is not observed in the absence of urea. Hence, we were able to evaluate the role of salt-screenable electrostatic interactions with different types of salts. Such examination would be much not possible if ionic denaturants, e.g., guanidinium chloride or thiocyanate, were used. Next, even in the presence of 9 M urea, the IF2 protein denatures at a relatively high melting temperature of 67 \({}^{\circ}\)C, and the protein remain reasonably stable. The major disadvantage and limitations of this method are the high nonphysiological concentrations of urea, its effect on water structure and activity, and the fact that urea can decompose and chemically react with proteins, leading to the formation of various chemical modifications in these proteins. For example, the decomposition of urea at high temperatures can generate isocyanic acid, which can react with the amino groups of lysine residues in proteins, forming carbamylated lysine residues [37]. Nevertheless, we observed high repeatability of thermal and cold denaturation. The high melting temperature of _Thermus_ IF2 in the presence of urea indicates high resistance against chemical denaturants and heat resistance. However, at room temperature, a large amount of the protein is unfolded as the protein undergoes cold denaturation. Therefore, a significantly high melting temperature does not guarantee high native state stability at room temperatures. Our study highlights the importance of studying denaturation processes at the lowest possible temperature, at approximately 2-5 \({}^{\circ}\)C in our case. In our study, we first examined how a salt screening of the electrostatic interactions affects cold denaturation. We found that three selected salts (Figure 2b; NaCl, NH4 Cl, and NaCl04) displayed specific effects on cold denaturation. Clearly, sodium chloride was very effective in suppressing cold denaturation, and we found that the other two salts were much less effective. To find out whether the suppression of the cold denaturation followed the Hofmeister series, we studied the effect of different salts (Figure 2c). In the presence of any of the salts, the transition temperature of the cold denaturation decreased.