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 (Ttrs, 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-tRNAfMet 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.

Cold unfolding of heat-responsive TRPV3

“…Cold unfolding of most proteins is di cult to detect when it occurs at temperatures well below water freezing. Therefore, except for Yfh1 and (Apo)-IscU, which may be the limited examples of natural proteins with an observable cold unfolding under (quasi) physiological conditions (31)(32), ad hoc mutations or denaturants such as urea or extreme pH values are usually used to destabilize globular proteins to increase a relatively lower melting temperature (T m, low ) in favor of detecting cold unfolding (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Similarly, although cold unfolding of membrane proteins is much more di cult to investigate structurally because of the effects of various membrane environments on protein stability, the K169A mutation could destabilize the closed state in favor of cold unfolding for detailed structural studies (20).…”

“…On the other hand, the relatively low melting temperature (T m, low ) can be available for detecting cold unfolding once ad hoc mutations or denaturants such as urea or extreme pH values destabilize globular proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Thereafter, it is tting to ask whether such a mutation could also increase the T m, low of the heat-responsive thermosensitive transient receptor potential (TRP) vanilloid 1-4 (TRPV1-4) channels for capturing cold unfolding (18).…”

“…For this reason, globular proteins were experimentally observed in only limited cases of cold unfolding by using nuclear magnetic resonance (NMR) and circular dichroism (CD) and differential scanning calorimetry (DSC). These proteins include metmyoglobin ( 1 ), staphylococcus nuclease ( 2 ), Streptomyces subtilisin inhibitor ( 3 – 4 ), ß-Lactoglobulin1 ( 5 ), barstar ( 6 ), bacterial cold shock protein ( 7 ), thioredoxin ( 8 ), L9 mutants ( 9 – 10 ), yeast Yfh1 and frataxin ( 11 – 14 ), HIV-1 protease monomer ( 15 ), E. coli IscU ( 16 ), and thermophilic multidomain initiation factor 2 ( 17 ). However, cold unfolding has never been reported for any membrane protein.…”