TAILIEUCHUNG - Báo cáo khoa học: Structural and thermodynamic analyses of Escherichia coli RNase HI variant with quintuple thermostabilizing mutations

A combination of five thermostabilizing mutations, Gly23fiAla, His62fiPro, Val74fiLeu, Lys95fiGly, and Asp134fiHis, has been shown to additively enhance the thermostability of Escherichia coliRN-ase HI [Akasako A, Haruki M, Oobatake M & Kanaya S (1995)Biochem-istry 34,8115–8122]. In this study, we determined the crystal structure of the protein with these mutations (5H-RNase HI) to analyze the effects of the mutations on the structure in detail. | ỊFEBS Journal Structural and thermodynamic analyses of Escherichia coli RNase HI variant with quintuple thermostabilizing mutations Mitsuru Haruki1 Masaki Tanaka2 Takehiko Motegi1 Takashi Tadokoro2 Yuichi Koga2 Kazufumi Takano2 and Shigenori Kanaya2 1 Department of Materials Chemistry and Engineering Nihon University Koriyama Japan 2 Department of Materialand Life Science Osaka University Suita Japan Keywords crystal structure quintuple thermostabilizing mutations RNase HI stability curve thermostability Correspondence M. Haruki Department of Materials Chemistry and Engineering College of Engineering Nihon University Koriyama Fukushima 963-8642 Japan Fax 81 24 956 8862 Tel 81 24 956 8794 E-mail haruki@ Received 30 April2007 revised 7 August 2007 accepted 11 September 2007 doi A combination of five thermostabilizing mutations Gly23 fi Ala His62 fi Pro Val74 fi Leu Lys95 fi Gly and Asp134 fi His has been shown to additively enhance the thermostability of Escherichia coli RNase HI Akasako A Haruki M Oobatake M Kanaya S 1995 Biochemistry 34 8115-8122 . In this study we determined the crystal structure of the protein with these mutations 5H-RNase HI to analyze the effects of the mutations on the structure in detail. The structures of the mutation sites were almost identical to those of the mutant proteins to which the mutations were individually introduced except for G23A for which the structure of the single mutant protein is not available. Moreover only slight changes in the backbone conformation of the protein were observed and the interactions of the side chains were almost conserved. These results indicate that these mutations almost independently affect the protein structure and are consistent with the fact that the thermostabiling effects of the mutations are cumulative. We also determined the protein stability curve describing the temperature dependence of the free energy of unfolding of 5H-RNase HI to .

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