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Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.

Okada J, Okamoto T, Mukaiyama A, Tadokoro T, You DJ, Chon H, Koga Y, Takano K, Kanaya S - BMC Evol. Biol. (2010)

Bottom Line: However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved.The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Material and Life Science, Osaka University, Suita, Japan.

ABSTRACT

Background: The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).

Results: To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.

Conclusions: These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

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GdnHCl concentration dependence of the apparent rate constant (ln kapp) of the unfolding of RNases H at 25°C. Open circles represent the data of Tm-RNase HII at pH 7.5; open triangles, that of Aa-RNase HII at pH 5.0; and closed squares, that of Sto-RNase HI at pH 3.0. The lines represent the fit of Eq. (6). Long dashed line represent the data of Tk-RNase HII and one-point dashed line represent Ec-RNase HI [19,31]. The red circle and blue triangles represents the ku(H2O) value obtained from urea-induced unfolding experiments with Ec-RNase HI [33] and Tt-RNase HI [21], respectively.
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Figure 3: GdnHCl concentration dependence of the apparent rate constant (ln kapp) of the unfolding of RNases H at 25°C. Open circles represent the data of Tm-RNase HII at pH 7.5; open triangles, that of Aa-RNase HII at pH 5.0; and closed squares, that of Sto-RNase HI at pH 3.0. The lines represent the fit of Eq. (6). Long dashed line represent the data of Tk-RNase HII and one-point dashed line represent Ec-RNase HI [19,31]. The red circle and blue triangles represents the ku(H2O) value obtained from urea-induced unfolding experiments with Ec-RNase HI [33] and Tt-RNase HI [21], respectively.

Mentions: Globular proteins usually unfold in the presence of GdnHCl. Unfolding takes less than 1 min in most small globular proteins. To measure such fast unfolding rates, a stopped-flow instrument is required. Ec-RNase HI is unfolded within 10 s by a final concentration of 3.0 M GdnHCl [31]. In contrast, the unfolding of some hyperstable proteins may take a few hours to several days. Pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf-PCP) is unfolded over a period of 1 day or more by a final concentration of 7.7 M GdnHCl [6], and the unfolding of Tk-RNase HII by a final concentration of 3.9 M GdnHCl requires 2 h [19]. The observed unfolding of all hyperstable proteins examined here is much slower than that of Ec-RNase HI in the presence of GdnHCl (Figures 2 and 3). For example, Tm-RNase HII is unfolded by 4.8 M GdnHCl within approximately 2 h; Aa-RNase HII is unfolded by 3.5 M GdnHCl within 1 h; and Sto-RNase HI is unfolded by 7.0 M GdnHCl within about 8 h. Our results suggest that such super slow unfolding in the presence of a chemical denaturant might be a common strategy for achieving higher stability and could be related to the adaptation of hyperthermophilic RNases H to higher temperatures.


Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.

Okada J, Okamoto T, Mukaiyama A, Tadokoro T, You DJ, Chon H, Koga Y, Takano K, Kanaya S - BMC Evol. Biol. (2010)

GdnHCl concentration dependence of the apparent rate constant (ln kapp) of the unfolding of RNases H at 25°C. Open circles represent the data of Tm-RNase HII at pH 7.5; open triangles, that of Aa-RNase HII at pH 5.0; and closed squares, that of Sto-RNase HI at pH 3.0. The lines represent the fit of Eq. (6). Long dashed line represent the data of Tk-RNase HII and one-point dashed line represent Ec-RNase HI [19,31]. The red circle and blue triangles represents the ku(H2O) value obtained from urea-induced unfolding experiments with Ec-RNase HI [33] and Tt-RNase HI [21], respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2927913&req=5

Figure 3: GdnHCl concentration dependence of the apparent rate constant (ln kapp) of the unfolding of RNases H at 25°C. Open circles represent the data of Tm-RNase HII at pH 7.5; open triangles, that of Aa-RNase HII at pH 5.0; and closed squares, that of Sto-RNase HI at pH 3.0. The lines represent the fit of Eq. (6). Long dashed line represent the data of Tk-RNase HII and one-point dashed line represent Ec-RNase HI [19,31]. The red circle and blue triangles represents the ku(H2O) value obtained from urea-induced unfolding experiments with Ec-RNase HI [33] and Tt-RNase HI [21], respectively.
Mentions: Globular proteins usually unfold in the presence of GdnHCl. Unfolding takes less than 1 min in most small globular proteins. To measure such fast unfolding rates, a stopped-flow instrument is required. Ec-RNase HI is unfolded within 10 s by a final concentration of 3.0 M GdnHCl [31]. In contrast, the unfolding of some hyperstable proteins may take a few hours to several days. Pyrrolidone carboxyl peptidase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf-PCP) is unfolded over a period of 1 day or more by a final concentration of 7.7 M GdnHCl [6], and the unfolding of Tk-RNase HII by a final concentration of 3.9 M GdnHCl requires 2 h [19]. The observed unfolding of all hyperstable proteins examined here is much slower than that of Ec-RNase HI in the presence of GdnHCl (Figures 2 and 3). For example, Tm-RNase HII is unfolded by 4.8 M GdnHCl within approximately 2 h; Aa-RNase HII is unfolded by 3.5 M GdnHCl within 1 h; and Sto-RNase HI is unfolded by 7.0 M GdnHCl within about 8 h. Our results suggest that such super slow unfolding in the presence of a chemical denaturant might be a common strategy for achieving higher stability and could be related to the adaptation of hyperthermophilic RNases H to higher temperatures.

Bottom Line: However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved.The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Material and Life Science, Osaka University, Suita, Japan.

ABSTRACT

Background: The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).

Results: To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.

Conclusions: These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.

Show MeSH
Related in: MedlinePlus