# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 2 Results
### Cold and Hot Denaturation Processes of IF2 Are Sensitive to Salts
(**c**) The thermal denaturation of IF2 in 0.1–2 M NH4 Cl, NaCl, CsCl, LiCl, and NaClO4 (\(T_{\text{trs, }\mathit{cold}}\) are labeled in gray and \(T_{\text{trs, }\mathit{hot}}\) in black). not achievable under the conditions of the study. The difference between Na\({}^{+}\) and NH\({}_{4}\)\({}^{+}\) chlorides indicates that the nature of the cation and its rank in the Hofmeister series affect the midpoint of cold denaturation. NH\({}_{4}\)\({}^{+}\) is a chaotropic cation, while sodium is a mild kosmotropic cation that can stabilize the native conformation of IF2. Such stabilization can be offset by a chaotropic anion. Therefore, in the following unfolding experiment, we used a combination of sodium and chaotropic anion-perchlorate. In the presence of 1.5 M NaClO\({}_{4}\), cold denaturation was again observed with \(T_{\mathit{cold}}\) = 11.6 \(\pm\) 0.2 \({}^{\circ}\)C. The temperature for thermal denaturation on the hot side decreased as expected for chaotropic anion, with \(T_{\mathit{trs, hot}}\) = 63.6 \(\pm\) 0.4 \({}^{\circ}\)C. One can conclude that cold and hot denaturation processes are dependent on the salt type and ionic strength. We systematically examined the thermal denaturation of IF2 in NH\({}_{4}\)Cl, NaCl, CsCl, LiCl, and NaClO\({}_{4}\) in the concentration range of 0.1-2 M (Figure 2c gray \(T_{\mathit{trs, cold}}\) and black \(T_{\mathit{trs, hot}}\)). In the presence of NH\({}_{4}\)Cl (Figure 2c), the thermal transition temperature \(T_{\mathit{trs, hot}}\) showed a hyperbolic increase. On the other hand, \(T_{\mathit{trs, cold}}\) displayed a linear decrease, indicating that IF2 becomes more stable and, hence, more folded at ambient temperature. Here, even though NH\({}_{4}\)\({}^{+}\) is a chaotropic cation, higher transition midpoint temperatures resulted in the stabilization of the protein at elevated temperatures. A similar increase in thermal denaturation on the hot side is visible in the presence of NaCl. Here, \(T_{\mathit{trs, hot}}\) increased dramatically; \(T_{\mathit{trs, hot}}\) increased by ca. 15 \({}^{\circ}\)C in the presence of 1 M salts and by 20 \({}^{\circ}\)C in 2 M NaCl. In the case of cold denaturation, \(T_{\mathit{trs, cold}}\) decreased to a small extent, by 3.8 \({}^{\circ}\)C, with 1 M NaCl, as can be seen from Figure 2c. However, cold denaturation was not observed because the CD signal was approximately halved compared with at 0.1 M NaCl. The possible reasons for this are discussed later in this paper. Thermal denaturation in the presence of CsCl mirrors the dependence found for NaCl (Figure 2c). Increasing the CsCl concentration significantly increased the transition temperature. At the same time, the threshold for cold denaturation shifted toward lower temperatures, and the cold denaturation process was not visible anymore above 0.5 M CsCl. LiCl and NaClO\({}_{4}\) salts were found to share similarities in terms of the concentration dependences of \(T_{\mathit{trs, hot}}\) and \(T_{\mathit{trs, cold}}\). Here, \(T_{\mathit{trs, hot}}\) decreased by 1.0 \(\pm\) 0.2 \({}^{\circ}\)C (2 M LiCl) or 5.2 \(\pm\) 0.2 \({}^{\circ}\)C (2 M NaClO\({}_{4}\)), which indicates a mild destabilization of native IF2 on the hot side. On the other hand, \(T_{\mathit{trs, cold}}\) decreased by 8.0 \(\pm\) 0.4 \({}^{\circ}\)C (at 2 M LiCl) and 9.8 \(\pm\) 0.2 \({}^{\circ}\)C (at 1.5 M NaClO\({}_{4}\)), indicating IF2 becomes more stable at ambient temperatures.