# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 3 Discussion
### Salt-Screenable Electrostatics of Interdomain Interactions
To analyze the internal organization of the domains of IF2, we performed frustration analysis [46; 47; 48]. The server that uses the so-called frustratometer algorithm classifies individual contacts based on their frustration index value. If a contact has a high frustration index it is relatively unfavorable compared with other possible contacts in that location. Conversely, if it has a low frustration index, it is relatively favorable compared with other possible contacts. Contacts with a frustration index that falls within a certain range are classified as \"minimally frustrated\", \"highly frustrated\", or \"neutral.\" A contact is considered \"minimally frustrated\" if its native energy is at the lower end of the distribution of decoy energies, indicating that most other amino acid pairs in that position would be unfavorable. A contact is considered \"highly frustrated\" if its native energy is at the other end of the  
Figure 5: The salt-concentration-dependent changes in enthalpy (**a**), heat capacity (**b**), and temperature of transition (**c**) for the G fragment (red) and C fragment (blue). (**d**) Distribution of surface electrostatic potential in the presence of 0.1 M salt (left) and 2 M salt (right). (**e**) The frustration index analysis performed by [43; 44; 45]. The color of the lines indicates whether a given contact is minimally (green) or highly (red) frustrated. The energy of a contact between two amino acids in a protein determines how favorable that interaction is. A contact is considered “minimally frustrated” if its energy is on the lower end of the possible range of energies for that type of contact. Conversely, a contact is considered “highly frustrated” if its energy is on the higher end of the range. For more details, please see the Discussion and Materials and Methods sections. Significant correlations and linear dependencies (\(\leq\)5% probability, yellow background in Table S1) are shown as stars (*). distribution of decoy energies, indicating that most other amino acid pairs at that location would be more favorable for folding than the native ones. Contacts that fall in between these limits are classified as \"neutral.\" Highly frustrated contacts can be problematic for protein stability and function because they may lead to local unfolding or misfolding of the protein. Identifying highly frustrated contacts can assist in understanding the structural and functional properties of a protein and can guide protein engineering efforts to improve stability and function. The analysis highlights that, while intradomain interactions show minimal frustration, the interdomain interaction between G domains, in particular, is highly frustrated (Figure 5e). Such a large frustration of interdomain interactions may have a biological significance due to the need for structural adaptation after binding to the ribosome. Upon binding, IF2 has to satisfy many contacts with other interacting sites on the ribosome and other molecules such as tRNA. The structurally encoded loss of the interdomain cooperativity may provide a suitable strategy to control the protein conformation by weakly stabilizing the frustrated internal network of electrostatic interactions. This control enables the protein to be compact in the absence of a ribosome. At the same time, upon binding and electrostatic shielding, the domains can now work more independently, which also allows securing contacts at distal sites.