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Journal of Biochemistry Advance Access published online on October 30, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn144
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© The authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Proline Effect on the Thermostability and Slow Unfolding of a Hyperthermophilic Protein

Kazufumi Takano1,2,*, Ryogo Higashi1, Jun Okada1, Atsushi Mukaiyama1, Takashi Tadokoro1, Yuichi Koga1 and Shigenori Kanaya1

1Department of Material and Life Science, Osaka University, Yamadaoka, Suita 565-0871, Japan
2CREST, Japan Science and Technology Agency (JST), Yamadaoka, Suita 565-0871, Japan

*Corresponding author: Prof. Kazufumi Takano, Department of Material and Life Science, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan, Tel/Fax: +81-6-6879-4157, E-mail: ktakano{at}mls.eng.osaka-u.ac.jp

Received August 19, 2008; Accepted October 18, 2008


  Abstract

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of {alpha}-helices. Proline residue at the N-terminal of an {alpha}-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of {alpha}-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an {alpha}-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.

Key Words: Folding, Hyperthermophilic protein, Proline residue, Ribonuclease HII, Stability


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