# 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  
_Thermus thermophilus_ IF2 consists of two parts that can be obtained after proteolytic cleavage [31]: a G fragment and a C fragment. Although the fragments have similar stabilities, two different denaturation pathways are represented. On the other hand, when the denaturation of domains is coupled, it can be described as a two-state process. For fragments displaying similar signals (as in our case, Figure 3a, similar secondary structure content) and independent denaturation, analysis of the thermal denaturation profiles can be misleading. We previously showed [28] that both fragments display similar thermal denaturation profiles; in the presence of 9 M urea, \(T_{\mathit{trs, hot}}\) for the G fragment is 67.6 \({}^{\circ}\)C and \(T_{\mathit{trs, hot}}\) for the C fragment is 71.6 \({}^{\circ}\)C. Hence, we assumed there are two denaturation pathways (Figure 3b). In one pathway, the denaturation starts from the C fragment, and in the second pathway, the unfolding starts from the G fragment. The observed denaturation process would correspond to the average signal, and the enthalpy and heat capacity would be much lower compared with the coupled all-or-nothing denaturation, where enthalpies and heat capacities add up. We conducted a thermodynamic analysis of IF2 in the presence of 0.1 M NaCl and found values that were too low, as was expected for such a large multidomain protein. To highlight the observed inconsistencies, the thermal profile of the CD signal of IF2 in 0.1 M NaCl was fitted by the Gibbs-Helmholtz equation with the parameters \(\Delta c_{p}\) 7.9 kJ/mol/K, \(\Delta H\) 212 kJ/mol, and \(T_{\mathit{trs, hot}}\) 69.1 \({}^{\circ}\)C,i.e., with a heat capacity value that is significantly smaller than expected for a 563 aa protein (7.9 vs. expected 33 kJ/K/mol) [11]. We assumed that the experimental curve could be decomposed according to independent unfolding of the G and C fragments, in which transitions are described by the same thermodynamic parameters (Figure 3c). Hence, the observed thermal profile reflects the sum of the unfolding of the independent fragments, with limited possibility to resolve individual fragment unfolding profiles and their corresponding thermodynamic parameters (Figure 3d,e). Mathematical Modeling--Effect of Individual Parameters and Transition Temperature (Figure 3f): We examined how the thermal profiles of independently unfolding fragments can be resolved and how they affect the apparent two-state character of thermal curves. The denaturation parameters for the C fragment were held constant (\(\Delta H\) 212 kJ/mol/K, \(\Delta c_{p}\) 7.9 kJ/mol, and \(T_{trs,\,hot}\) 342.2 K) while the parameters for the G fragment were changed as indicated in Figure 3f (variable \(T_{trs,\,hot}\), \(\Delta H\) 212 kJ/mol/K, \(\Delta c_{p}\) 7.9 kJ/mol). A difference of 10 \({}^{\circ}\)C in \(T_{trs,\,hot}\) between fragments did not allow resolving individual unfolding transitions. Under the parameters of the study, a difference of at least 20 \({}^{\circ}\)C was needed to resolve transitions in both the hot and cold sides. From a technical viewpoint, multistate behavior on the hot side was more evident due to the presence of post- and pretransition baselines. This situation was reversed when \(T_{trs,\,hot}\) shifted by at least 20 \({}^{\circ}\)C toward a higher temperature; here, the cold denaturation profile exhibited clear post- and pretransition baselines and, hence, fragment unfolding became visible. To conclude, even with a 15 \({}^{\circ}\)C difference between \(T_{trs,\,hot}\) of the fragments was not sufficient to allow distinguishing between individual transitions. Enthalpy (Figure 3g): A decrease in the enthalpy of the G fragment would result in an evident fingerprint of multistate unfolding. Importantly, the simulated enthalpies (112 and 12 kJ/mol) were quite low and on the small side of the usual enthalpy range.