# 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
In summary, simulations of independent thermal denaturation transitions were useful for achieving resolution based on our experimental method. Our simulations indicate the highly limited resolution of the denaturation curves and thermodynamic parameters for the full-length protein because of the significant overlap in the denaturation curves for both fragments. Hence, instead of studying the full-length protein, we focused on the individual fragments: the G and C fragments. Salt-Dependent Thermal Denaturation of Individual Fragments Reveals Differences in Salt-Screenable Electrostatic Interactions  
As indicated by our simulations, it is important to dissect multidomain proteins and separately analyze their fragments. To this end, we prepared and isolated individual fragments of IF2 (see [28]). Thermal denaturation using circular dichroism in the far-UV region showed that both fragments undergo cold and hot denaturation (Figure 3a). Using nonlinear regression analysis of the two-state model, we were able to obtain the thermodynamic values \(\Delta H\), \(\Delta c_{p}\), and \(T_{trs,\,hot/cold}\) for the individual transitions. Due to cold and hot denaturation, all parameters could be obtained with reasonable accuracy. We found that \(\Delta H_{app}\) and \(\Delta c_{p,\,app}\) of the individual fragments are similar regarding their full-length IF2. We wondered whether the stability of individual fragments would also display the same salt and ionic strength concentration dependence. To this end, the thermal denaturation experiments were conducted for both fragments and analyzed. The thermodynamic data are shown in Figure 4a-d ac (G fragment in red and C fragment in blue) and are in agreement with the values found for other proteins [11]. In the presence of NH\({}_{4}\)Cl, the unfolding enthalpy for both G and C fragments decreased. However, the salt dependence of the G fragment, was slightly higher. The same was true for \(\Delta c_{p,\,app}\), which decreases significantly at high NH\({}_{4}\)Cl concentrations, from 8.9 to 4.5 kJ/mol/K at 2 M. Again, the \(\Delta c_{p,\,app}\) was slightly more salt-sensitive for the G fragment. In 0.1 M NH\({}_{4}\)Cl, the transition temperature was 65.0 \({}^{\circ}\)C for the G fragment and 70.0 \({}^{\circ}\)C for the C fragment. As part of the full-length IF2, the \(T_{trs,\,hot}\) fragments were separated by less than 5 \({}^{\circ}\)C; hence, we could not resolve the individual transitions. \(T_{trs,\,hot}\) for both fragments were dependent on NH\({}_{4}\)Cl, and we observed that \(T_{trs,\,hot}\) was more salt-dependent for the C fragment than the G fragment. A much larger difference and high salt sensitivity were found for NaCl; again, the unfolding enthalpy and heat capacity change decreased at high salt concentrations of NaCl. This change was even more pronounced for the G fragment when \(\Delta H_{app}\) reduced from 212 to 50 kJ/mol at 2 M NaCl. At the same time, the C fragment \(\Delta H_{app}\) decreased from 212 kJ/mol at 0.1 M NaCl to 138.5 kJ/mol at 2 M NaCl. The salt-induced change in heat capacity is clearly visible for both fragments, with \(\Delta c_{p,\,app}\) reduced to 4 kJ/mol/K for the C fragment and 2 kJ/mol for the G fragment. Again, the slope is higher for the G fragment, indicating higher salt sensitivity. The transition temperature in 0.1 M NaCl is 65.7 \({}^{\circ}\)C for the G fragment and 71.5 \({}^{\circ}\)C for the C fragment. The difference between \(T_{trs,\,hot}\) for both fragments increases dramatically due to a significant drop in \(T_{trs,\,hot}\) of the G fragment at a high salt concentration, highlighting the high salt sensitivity of the G fragment. At 2 M NaCl, \(T_{trs,\,hot}\) is 56.4 \({}^{\circ}\)C for the G fragment and 85.1 \({}^{\circ}\)C for the C fragment, which are substantially different and could, therefore, be potentially observable in full-length IF2.