# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 2 Results
### Limitations to the Analysis of Thermal Denaturation of Two Overlapping Independent Fragments
Our mathematical simulations (see Section 2.2) were performed with one variable parameter (e.g., \(T_{trs,\,hot}\)), while the other thermodynamic parameters remained fixed (e.g., \(\Delta H\), \(\Delta c_{p}\)). Our analysis of experimental data clearly shows that all parameters are salt-dependent. Hence, they may compromise the ability to resolve individual transitions even if the difference in \(T_{trs,\,hot}\) is significant enough for a certain combination of the thermodynamic parameters. Using experimentally obtained thermodynamic parameters for individual domains at 2 M NaCl, we calculated the profiles of the individual denaturation curves of G and C fragments. Again, we used temperature steps and extracted the noise from experiments. After the simple addition of the calculated denaturation curves of fragments, we obtained a hypothetical denaturation curve for full IF2. Then, the calculated full IF2 denturation curve was analyzed and subjected to visual assessment. We found that even a 28 \({}^{\circ}\)C difference in transition temperatures for the G and C fragments is insufficient when the denaturation enthalpies and heat capacities are very low, as was observed with 2 M NaCl. In summary, in the presence of NaCl and CsCl, the G fragment displays nonlinear salt dependence of transition temperatures, while the C fragment displays linear dependences. Individual denaturation processes for G/C domains could not be observed in the full-length protein because the small magnitude of enthalpy and heat capacity changes further decreased the resolution. Figure 4: The thermodynamic parameters obtained from nonlinear regression of the cold and hot denaturation data: \(\Delta H\) **(a)**; \(\Delta\sigma_{p}\) **(b)**; \(T_{ts, hot}\) **(c)**; and **(d) \(T_{ts, cold}\)** for G fragment (red) and C fragment (blue) in the presence of 9 M urea, and 0.1-2 M salts (NH4 Cl; NaCl; CsCl; LiCl; and NaClO4). Data were analyzed using linear regression analysis, and Pearson correlation coefficients are listed in Supplementary Table S1. In addition, the table contains the percentage of the probability \(Prob_{\rm N}\) (\(1r\,|\geq r_{0}\)) that the correlation coefficient was observed for uncorrelated variables containing \(N\) data pairs (see [33]). The quadratic fit was applied when the residuals from linear regression analysis displayed significant trends, deviations, and/or serial correlations. The major function of the regression fits was to guide the eye and quantify the salt sensitivity of the given parameter. For residual plots, see [34; 35]. We found that in the presence of CsCl, the differences between G and C fragments were often very subtle and with similar values for \(\Delta H_{app}\) and \(\Delta c_{p,\,app}\). Decreases in \(\Delta H_{app}\) were observed at high salt concentration, with only a slight difference in the salt dependence slope for G and C fragments. As was found for other salts, \(\Delta c_{p,\,app}\) decreases as the salt concentration is increased. Here, \(\Delta c_{p,\,app}\) for G and C fragments demonstrated nearly equal salt sensitivity and slope compared with variations in salt. In 0.1 M CsCl, the transition temperature was 65.5 \({}^{\circ}\)C for the G fragment and 71.1 \({}^{\circ}\)C for the C fragment. Similarly, as for NaCl dependence, the difference between \(T_{ts,\,hot}\) for both fragments increased dramatically due to the significant nonlinear drop in \(T_{ts,\,hot}\) of the G fragment at high salt concentration, hence highlighting the high salt sensitivity of the G fragment. At 2 M CsCl, \(T_{ts,\,hot}\) was 50.5 \({}^{\circ}\)C for the G fragment and 79.0 \({}^{\circ}\)C for the C fragment, which represents a substantial difference that could be potentially observable in full-length IF2. Again, as in the case of 2 M NaCl, we simulated the individual unfolding transitions for the G and C fragments. Here, similar to our previous observations, the resolution was compromised due to a large decrease in enthalpy and heat capacity.