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. 2011 Jan 19;6(1):e16226.
doi: 10.1371/journal.pone.0016226.

Stabilization by fusion to the C-terminus of hyperthermophile Sulfolobus tokodaii RNase HI: a possibility of protein stabilization tag

Kazufumi Takano  1 , Tomohiro Okamoto, Jun Okada, Shun-ichi Tanaka, Clement Angkawidjaja, Yuichi Koga, Shigenori Kanaya
Affiliations

Stabilization by fusion to the C-terminus of hyperthermophile Sulfolobus tokodaii RNase HI: a possibility of protein stabilization tag

Kazufumi Takano et al. PLoS One. .

Abstract

RNase HI from the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) is stabilized by its C-terminal residues. In this work, the stabilization effect of the Sto-RNase HI C-terminal residues was investigated in detail by thermodynamic measurements of the stability of variants lacking the disulfide bond (C58/145A), or the six C-terminal residues (ΔC6) and by structural analysis of ΔC6. The results showed that the C-terminal does not affect overall structure and stabilization is caused by local interactions of the C-terminal, suggesting that the C-terminal residues could be used as a "stabilization tag." The Sto-RNase HI C-terminal residues (-IGCIILT) were introduced as a tag on three proteins. Each chimeric protein was more stable than its wild-type protein. These results suggested the possibility of a simple stabilization technique using a stabilization tag such as Sto-RNase HI C-terminal residues.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Crystal structure of wild-type Sto-RNase HI.
C-terminal seven residues (cyan); hydrophobic side-chains (blue); hydrogen bonds (thin red lines); and disulfide bond (thick red line).
Figure 2
Figure 2. CD spectra and crystal structure of Sto-RNase HI variants.
(A) CD spectra of wild-type (solid line), C58/145A (dashed line), and ΔC6 (dot-dashed line) Sto-RNase HI. (B) Crystal structure of ΔC6 Sto-RNase HI. (C) Crystal structure of wild-type Sto-RNase HI. The C-terminal seven residues are in cyan.
Figure 3
Figure 3. GdnHCl-induced equilibrium unfolding curves and thermodynamic stability profiles (temperature dependence of ΔG(H2O)) of Sto-RNase HI.
Wild-type (solid line and circles), C58/145A (dashed line and triangles), and ΔC6 (dot-dashed line and squares). (A) GdnHCl-induced equilibrium unfolding at 20°C. The apparent fraction of unfolded protein is shown as a function of GdnHCl concentration. Lines are best fits to a two-state equation. (B) Thermodynamic stability profiles. Closed symbols are the Tm value from the heat-induced unfolding experiment . Lines represent the fit of Eq. (3) using both equilibrium and heat-induced unfolding data.
Figure 4
Figure 4. GdnHCl-induced kinetic unfolding curves of Sto-RNase HI.
Lines represent the fit of Eq. (4). (A) C58/145A. Curve represents the unfolding trace to a final concentration of 5.8 M GdnHCl. (B) ΔC6. Curve represents the unfolding trace to a final concentration of 5.0 M GdnHCl.
Figure 5
Figure 5. Crystal structure of So-RNase HI, Ec-RNase HI, and Sto-esterase.
(A) So-RNase HI, (B) Ec-RNase HI and (C) Sto-esterase.
Figure 6
Figure 6. SDS-PAGE and heat-induced unfolding curves of chimeric proteins.
(A) SDS-PAGE. Lanes 1, 3, 5, are a low-molecular weight marker kit (GE Healthcare). Lanes 2, 4, 6 are purified chimeric So-RNase HI, Ec-RNase HI and Sto-esterase. (B) Heat-induced unfolding. The apparent fraction of unfolded protein is shown as a function of temperature. Curves 1, 2 and 3 represent the unfolding traces of chimeric So-RNase HI (closed circles), Ec-RNase HI (open circles) and Sto-esterase (cross). Lines are best fits to a two-state equation.
See this image and copyright information in PMC

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