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Report No.

Seven cysteine-deficient mutants depict the interplay between thermal and chemical stabilities of individual cysteine residues in mitogen-activated protein kinase c-Jun N-terminal kinase 1

Nakaniwa, Tetsuko*; Fukata, Harumi*; Inoue, Tatsuya*; Goda, Masaki*; Nakai, Ryoko*; Kirii, Yasuyuki*; Adachi, Motoyasu; Tamada, Taro; Segawa, Shinichi*; Kuroki, Ryota; Tada, Toshiji*; Kinoshita, Takayoshi*

Protein kinase is a vital drug target for the treatment of a wide range of diseases. To investigate the effect of cysteine mutation on the function, stability and structure of kinase, free cysteines of c-Jun N-terminal kinase 1 (JNK1) were systematically removed by mutation. Two cysteine-destructed mutants in which three (M3) and seven (M7) cysteine residues are removed, yielded about 5 and 2 times than wild type JNK-1 (M0). SDS PAGE analysis showed that the aggregation was less in the case of M3 and M7. Thermal unfolding experiment of M0, M3 and M7 using by differential scanning calorimetry proceeded at least three state unfolding. Crystal structure of the M3 mutant was determined to 2.6 ${AA}$ resolution, which was identical to that of the wild-type. Consequently, due to the highest yield, its improved stability against aggregation and its structural similarity to the wild type, the M3 mutant is suitable for the use of further characterization of its function and structure.



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Category:Biochemistry & Molecular Biology



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