# Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2
## 1 Introduction  
Thermophilic bacterial proteins and enzymes are attractive to the academic sector and for industrial applications [1, 2]. Thermophilic proteins and enzymes display high thermal stability and significant activity at elevated temperatures. The high stability and activity of thermophilic proteins at elevated temperatures enable conducting experiments and observing reactions that do not occur at room temperature (e.g., polymerase chain reaction). The molecular mechanism of thermostability has been intensively investigated [3, 4, 5, 6, 7]. In quantitative terms, it is expressed by a protein stability curve that describes how the free energy of the denaturation, \(\Delta G\), is dependent on temperature and how the denaturation enthalpy and entropy change over an extensive temperature range. In the case of proteins, denaturation enthalpy is strongly temperature-dependent due to the high heat capacity change in the process. A significant positive change in heat capacity has an interesting consequence for the depiction of protein stability as a curve, which adopts a parabolic shape. Hence, two different denaturation processes can be observed: one on the hot side and one on the cold side--so-called cold denaturation. The molecular origins of colddenaturation lie in the temperature-dependent hydration of hydrophobic groups, which becomes more favorable at low temperatures [8]. Cold denaturation is not often observed, mainly due to two interrelated reasons. First, cold denaturation usually occurs at temperatures well below the experimental window of spectroscopic/calorimetric techniques. To observe cold denaturation at ambient temperatures, the protein stability curve, \(\Delta G\) vs. \(T\), should be shifted horizontally to higher temperatures, indicating a higher hot denaturation transition temperature. Thus, thermophilic proteins can be good candidates for observing cold denaturation. However, the presence of residual structure in the unfolded state may decrease heat capacity [9; 10] and, hence, reduce the curvature of the protein stability curve, which then shifts the threshold for cold denaturation to temperatures that are not readily achievable. Second, the changes in heat capacity are often not sufficiently high to allow observation of cold denaturation. As heat capacity changes are correlated with the number of residues [11] or, more precisely, with the size of the cooperative unit, cold denaturation should be reversible and readily detectable for larger proteins. On the other hand, larger proteins are often arranged in domains (beads on a string or discontinuous domains) that may independently fold/unfold, diminishing the size of the cooperative unit and, hence, the magnitude of the heat capacity change. Taken together, there are only limited cases under which cold denaturation can be observed. Translation initiation factor 2 (IF2, Figure 1) is among the largest nonribosomal GTPase involved in initiating protein biosynthesis in the cell. It belongs to the family of structurally related enzymes catalyzing GTP hydrolysis during the early processes of mRNA translation. IF2 plays a central role in translation initiation via the specific selection of fMet-tRNA(Met). IF2 specific selection is based on recognition of the N-formyl-methionine and aminoacyl acceptor stem of fMet-tRNA(Met, 12; 13) and significantly faster association of the 50S subunit and correctly assembled 30S complex, which contains all functional components for successful translation initiation. IF2 also participates in other cellular processes, such as transcription [14], the protein export reaction [15], and the prevention of protein aggregation [16], and it functions as a cellular metabolic sensor that toggles between an active, GTP-bound form under normal conditions and an inactive form with bound ppGpp--a nucleotide messenger produced in bacteria during their starvation [17].