# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 3 Discussion
### Salt-Screenable Electrostatics of Interdomain Interactions  
We observed an apparent decrease in the denaturation enthalpy in the presence of all salts (Figure 5a, negative slopes for \(\Delta\Delta H/\Delta\)_salt_). A negative slope indicates that electrostatic interactions in the native state actually contribute favorably, in a highly enthalpic manner, to the stability of IF2. Furthermore, the presence of salt screens out favorable interactions. As mentioned above, the decrease in enthalpy is also accompanied by the loss of heat capacity change, which can be due to changes on the native side, for example, in the size of the cooperative units or changes in the compactness of the unfolded state. Based on the analysis of the CD profiles of thermal transition, we can exclude the formation of an even more profound residual structure in the unfolded state. Therefore, the loss of enthalpy and heat capacity change is likely due to a decrease in the cooperative unit. A plausible explanation for this is the loss of folding cooperativity between the individual domains in the G and C fragments. In the case of the G fragment, the G1 domain is clearly separated from the other G2/G3 domains; therefore, G1 can be seen as a candidate for an autonomous, thermodynamically stable, folded unit. For the C fragment, we can identify two C1/C2 domains, which are also separated in space; therefore, it is plausible that the domains can independently denature under some conditions, e.g., in the presence of the salts. We observed that the changes in denaturation enthalpy and heat capacity are more salt-sensitive (higher absolute slope values) in the domains of the G fragment than for the C fragment. This difference may arise from the different number of domains in the G and C fragments, and from inherent differences in the surface electrostatics. Interestingly, with the exception of perchlorate, most salts increase the melting temperature, indicating stabilization due to salts is entropic. A favorable entropic term may be attributed to the native state increase in the entropy upon losing the domain-domain interactions. As opposed to the C domains, G domains consist of several clusters of charged residues (Figure 5). Again, a higher number of positively charged residues may also result in higher salt sensitivity and, hence, higher absolute values of the slopes of \(\Delta H\) vs. salt concentration. In the case of perchlorate, the native state is destabilized, as indicated by decreases in both melting temperature and denaturation enthalpy. However, cold denaturation is not shifted toward higher temperatures, which might be expected in the case of the native state destabilization relative to the unfolded state. The change in the heat capacity of the C fragment is slightly positive, in contrast to the G fragment, where perchlorate dramatically decreases heat capacity. The type of anions appears to have a much greater influence when one compares sodium chloride with perchlorate. This might be due to the screening efficiency of the localized exposed positive charge in the C fragment (Figure 5d). The observed slopes of thermodynamic parameters obtained for different salts (e.g., \(\Delta\Delta H/\Delta\)[salt]) are specific for a given chloride salt and may be correlated with some of the physicochemical properties of the cation. Similar correlations were found in different studies of proteins in the presence of salts of the [PERSON] series [42; 43; 44]. In our case, we could not find any evidence for a robust correlation between the salt-specific values of the slopes and ion properties such as hydration entropy, hydration number, charge density, polarizability, and viscosity B coefficient (see Figures S6-S17, Supplementary Materials, which includes also pairwise correlations between thermodynamic parameters of domains). The lack of a clear correlation can be possibly attributed to the complexity of the effects of cations on stability and resolvable transitions and the multistate denaturation process, which we addressed in this work.