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Nakaniwa, Tetsuko*; Fukata, Harumi*; Inoue, Tatsuya*; Goda, Masaki*; Nakai, Ryoko*; Kirii, Yasuyuki*; Adachi, Motoyasu; Tamada, Taro; Segawa, Shinichi*; Kuroki, Ryota; et al.
Biochemistry, 51(42), p.8410 - 8421, 2012/10
Times Cited Count:18 Percentile:42.99(Biochemistry & Molecular Biology)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 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.
Nakaniwa, Tetsuko*; Fukata, Harumi*; Inoue, Tatsuya*; Kinoshita, Takayoshi*; Adachi, Motoyasu; Tamada, Taro; Kuroki, Ryota; Tada, Toshiji*
no journal, ,
JNK1 is a MAP kinase responsible for response of stress. JNK1 has 4 and 3 cysteine residues in embedded region and at molecular surface, respectively. Those cysteine residues would cause inactivation and aggregation of the molecule. Based of the analysis of packing in crystal of isozyme of JNK1, we found more salt bridge and hydrogen bonding interactions on the interface. In this study, we focus on the two cysteine residues and introduced modification into M3 mutant previously reported.