Abstract
Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from Halobacterium salinarum ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of E. coli CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0–3 M KCl, while the structure of E. coli CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the E. coli enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.
Highlights
Understanding the effects that environmental factors have on a protein’s structure is a difficult task
Potassium was observed to induce the greatest change in NRC-1 CysRS structure (65% increase in signal) which fits with its potassium rich cellular environment [25,26,27]
The large change in fluorescence emission spectra of NRC-1 CysRS in high salt compared to the E. coli version suggests that the halophilic version is more structurally sensitive to its ionic environment (Figure 3)
Summary
Understanding the effects that environmental factors have on a protein’s structure is a difficult task. Electrostatic interactions between the protein’s polar residues are able to form salt bridges and hydrogen bonds that essentially lock the protein into a particular configuration. Chaperonins play a major role in protein folding and structure [1,2,3,4]. They have the ability to change the protein’s local environment and change the way the protein folds. Thermophiles, whose proteins have more hydrophobic residues and increased ionic interactions over nonthermophilic proteins, take advantage of the more favorable salt bridge interactions possible due to higher temperatures in order to fold their proteins [11,12,13]. Higher temperatures are a stabilizing force for thermophilic proteins and encourages folding with very slow unfolding pathways [12]
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