# 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  
Circular dichroism in the far-UV region is a highly sensitive technique for studying the conformational stability of proteins. This is due to the strong absorption of the peptide group and the high sensitivity of electron transitions n\(\rightarrow\)\(\pi^{*}\) (at 222 nm) and \(\pi\)\(\rightarrow\)\(\pi\)\({}^{*}\) (at 208 nm) to the protein secondary structure [30]. Circular dichroism at 222 nm was used to monitor the thermal unfolding of IF2 in the presence of 0.1 M NaCl and a phosphate buffer (Figure 2a). At the initial stages of thermal denaturation at 4 \({}^{\circ}\)C, the ellipticity was observed to have a sigmoidal dependence on temperature. Then, at 30 \({}^{\circ}\)C, the ellipticity remained constant until the temperature reached 60 \({}^{\circ}\)C and further increased; again, a sigmoidal-like unfolding transition could be observed. We attributed both sigmoidal processes to cold and hot denaturation (see labels \(T_{\textit{trs, cold}}\) and \(T_{\textit{trs, hot}}\) in Figure 2a). Hence, at ambient temperatures (ca. 20 \({}^{\circ}\)C), the thermophilic multidomain protein IF2 was largely unfolded (ca. 50%). To obtain the transition midpoint temperatures in a model-independent way, we applied an approach based on the first derivative of the experimental curves. Upon derivation, two extrema were observed: a minimum, which corresponds to the midpoint of cold denaturation; and a maximum, which corresponds to the midpoint of hot denaturation. Both denaturing transitions were cooperative, and the temperatures corresponding to cold and hot denaturation were \(T_{\textit{trs, cold}}\) 18 \(\pm\) 0.4 \({}^{\circ}\)C and \(T_{\textit{trs, hot}}\) 67.4 \(\pm\) 0.2 \({}^{\circ}\)C.  
One of the possible mechanisms underlying the thermal adaptation of thermophilic proteins is based on the optimization of electrostatic interactions on the protein surface. The role of electrostatic interactions in determining protein stability can be studied by examining the dependence of protein stability on the concentration different salt types. To understand the role of electrostatic interactions in the stability of thermophilic IF2, we first conducted experiments using selected salts at a very high ionic strength: 1.5 M (Figure 2b). Figure 2b shows the thermal denaturation of IF2 in the presence of 1.5 M NaCl, NH4 Cl, and NaClO4, plotted as unfolded fraction (\(f_{\text{u}}\)) vs. temperature (\(T\)). This combination allows us to compare the role of cations of different ranks in the Hofmeister series (Na+ versus NH4+, both chlorides) and different anions (Cl\({}^{-}\) versus ClO4\({}^{-}\), Na+). In the presence of NH4 Cl, only minor shifts in \(T_{\text{tot}}\) toward higher temperatures at higher salt concentrations and a slight shift in \(T_{\text{cold}}\) to lower values were observed. In the presence of NaCl, we found that thermal denaturation on the hot side shifted toward higher temperatures at higher salt concentrations. Interestingly, at 1.5 M NaCl, cold denaturation of IF2 was not observed due to a significant shift in the threshold toward lower temperatures that were  
Figure 2: (**a**) Thermal unfolding of IF2 in the presence of 0.1 M NaCl, 9 M urea in the buffer monitored by circular dichroism in the far – UV region at 222 nm. (**b**) Thermal denaturation of IF2 in the presence of 1.5 M NaCl, NH4 Cl, and NaClO4 plotted as an unfolded fraction (\(f_{\text{u}}\)) vs. temperature (T). For the corresponding CD spectra of IF2 shown in (**a**) at 4, 40, and 95 \({}^{\circ}\)C, see Figure S1, Supplementary Materials. Data were normalized based on the averaged signal measured when the protein is folded (between 35 and 45 \({}^{\circ}\)C) and to the U state based on the average signal measured when the protein is unfolded (\(>\)90 \({}^{\circ}\)C, for fitting see Figure S2, Supplementary Materials).