Salt-Specific Suppression of the Cold Denaturation of Thermophilic Multidomain Initiation Factor 2

Abstract

Thermophilic proteins and enzymes are attractive for use in industrial applications due to their resistance against heat and denaturants. Here, we report on a thermophilic protein that is stable at high temperatures (T_{trs, hot} 67 °C) but undergoes significant unfolding at room temperature due to cold denaturation. Little is known about the cold denaturation of thermophilic proteins, although it can significantly limit their applications. We investigated the cold denaturation of thermophilic multidomain protein translation initiation factor 2 (IF2) from Thermus thermophilus. IF2 is a GTPase that binds to ribosomal subunits and initiator fMet-tRNA^fMet during the initiation of protein biosynthesis. In the presence of 9 M urea, measurements in the far-UV region by circular dichroism were used to capture details about the secondary structure of full-length IF2 protein and its domains during cold and hot denaturation. Cold denaturation can be suppressed by salt, depending on the type, due to the decreased heat capacity. Thermodynamic analysis and mathematical modeling of the denaturation process showed that salts reduce the cooperativity of denaturation of the IF2 domains, which might be associated with the high frustration between domains. This characteristic of high interdomain frustration may be the key to satisfying numerous diverse contacts with ribosomal subunits, translation factors, and tRNA.

Keywords: cold denaturation; domain cooperativity; electrostatic interactions; initiation factor 2; thermodynamic stability.

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 (≤5% probability, yellow background in Table S1) are shown as stars (*).

Figure 1

The division (upper part) of translation initiation factor 2 from Thermus thermophilus into the G fragment (red), consisting of G1 and G2/3 domains, and the C fragment (blue) consisting of C1 and C2; cryo-EM model (lower part) of ribosome-bound IF2 prepared based on the structure in PDB [5LMV], with binding partners (IF1, tRNA, mRNA, and IF3) shown as colored molecular surfaces.

Figure 2

(a) Thermal unfolding of IF2 in the presence of 0.1 M NaCl, 9 M urea in the buffer monitored by circular dichroism in the far−UV region at 222 nm. (b) Thermal denaturation of IF2 in the presence of 1.5 M NaCl, NH₄Cl, and NaClO₄ plotted as an unfolded fraction (f_u) vs. temperature (T). For the corresponding CD spectra of IF2 shown in (a) at 4, 40, and 95 °C, see Figure S1, Supplementary Materials. Data were normalized based on the averaged signal measured when the protein is folded (between 35 and 45 °C) and to the U state based on the average signal measured when the protein is unfolded (>90 °C, for fitting see Figure S2, Supplementary Materials). (c) The thermal denaturation of IF2 in 0.1–2 M NH₄Cl, NaCl, CsCl, LiCl, and NaClO₄ (T_{trs, cold} are labeled in gray and T_{trs, hot} in black).

Figure 3

(a) The thermal denaturation of the G fragment (red), C fragment (blue), and full-length IF2 (black) measured by circular dichroism at 222 nm. (b) Two denaturation pathways of IF2: in one pathway, denaturation starts with the C fragment, and in the second pathway, denaturation starts with the G fragment. When the fragments have equal stability and are independent, then both pathways are present. (c) Independent unfolding of G and C fragments in which transitions are described by the same thermodynamic parameters. (d) Simulation of unfolding transition of IF2 in the case when the denaturation of both G and C fragments is coupled (dashed line) and independent (solid line) and measured data (gray dots). In simulations, data are described by the same parameters. (e) Simulated stability curves for independent G/C fragments (solid red and blue line) and coupled fragments (dashed black line) described by the same thermodynamic parameters as in (d). Simulation of changes in the thermal unfolding profile when the denaturation parameters for the C fragment are held constant (ΔH 212 kJ/mol/K, Δc_p 7.9 kJ/mol, T_{trs, hot} 342.2 K) while one of the parameters for the G fragment is varied (f) variable T_{trs, hot}, ΔH 212 kJ/mol/K, and Δc_p 7.9 kJ/mol and (g) T_{trs, hot} 342.2 K, variable ΔH, and Δc_p 7.9 kJ/mol. (h) Simulated effect of changes in the values of the heat capacity for the G fragment with the other values held constant. See also Figures S3–S5, Supplementary Materials.

Figure 4

The thermodynamic parameters obtained from nonlinear regression of the cold and hot denaturation data: ΔH (a); Δc_p (b); T_{trs, hot} (c); and (d) T_{trs, cold} for G fragment (red) and C fragment (blue) in the presence of 9 M urea, and 0.1–2 M salts (NH₄Cl; NaCl; CsCl; LiCl; and NaClO₄). Data were analyzed using linear regression analysis, and Pearson correlation coefficients are listed in Supplementary Table S1. In addition, the table contains the percentage of the probability Prob_N (|r| ≥ r₀) that the correlation coefficient was observed for uncorrelated variables containing N data pairs (see [33]). The quadratic fit was applied when the residuals from linear regression analysis displayed significant trends, deviations, and/or serial correlations. The major function of the regression fits was to guide the eye and quantify the salt sensitivity of the given parameter. For residual plots, see [34,35].