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Journal of Biochemistry Advance Access published online on August 30, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm157
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© 2007 The Japanese Biochemical Society

Protein thermostabilization requires a fine-tuned placement of surface charged residues

Dong-Ju You1, Satoshi Fukuchi2, Ken Nishikawa2, Yuichi Koga1, Kazufumi Takano1,3,* and Shigenori Kanaya1

1Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan
3CREST, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

*Corresponding author, Prof. Kazufumi Takano, E-mail address of the corresponding author: ktakano{at}mls.eng.osaka-u.ac.jp Tel/Fax of the corresponding author: +81-6-6879-4157

Received June 14, 2007; Accepted July 23, 2007


  Abstract

Using the information from the genome projects, recent comparative studies of thermostable proteins have revealed a certain trend of amino acid composition in which polar residues are scarce and charged residues are rich on the protein surface. To clarify experimentally the effect of the amino acid composition of surface residues on the thermostability of Escherichia coli Ribonuclease HI (RNase HI), we constructed six variants in which five to eleven polar residues were replaced by charged residues (5C, 7Ca, 7Cb, 9Ca, 9Cb, and 11C). The thermal denaturation experiments indicated that all of the variant proteins are 3.2° to 10.1°C in Tm less stable than the wild proteins. The crystal structures of resultant protein variants 7Ca, 7Cb, 9Ca, and 11C closely resemble that of E. coli RNase HI in their global fold, and several different hydrogen bonding and ion-pair interactions are formed by the mutations. Comparison of the crystal structures of these variant proteins with that of E. coli RNase HI reveals that thermal destabilization is apparently related to electrostatic repulsion of the charged residues with neighbors. This result suggests that charged residues of natural thermostable proteins are strictly posted on the surface with optimal interactions and without repulsive interactions.

Key Words: amino acid composition, genome information, RNase HI, surface-charge residue, thermostability


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