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    Please use this identifier to cite or link to this item: https://ir.cnu.edu.tw/handle/310902800/25230

    標題: Site-Saturation Mutagenesis of Leucine 134 of Bacillus licheniformis Nucleotide Exchange Factor GrpE Reveals the Importance of this Residue to the Co-chaperone Activity
    作者: Min-Guan Lin
    Bo-En Chen
    Wan-Chi Liang
    Wei-Mou Chou
    Jiau-Hua Chen
    Lih-Ying Kuo
    Long-Liu Lin
    貢獻者: 食品科技系
    關鍵字: Bacillus licheniformis
    Site-saturation mutagenesis
    Co-chaperone activity
    Temperature-induced denaturation
    日期: 2010-06
    上傳時間: 2012-05-22 13:53:31 (UTC+8)
    摘要: To elucidate the role of leucine 134 of Bacillus licheniformis nucleotide exchange factor (BlGrpE), site-saturation mutagenesis was employed to generate all possible replacements for this residue. Wild-type and mutant proteins were purified by nickel-chelated chromatography and had a molecular mass of approximately 34.5 kDa. As compared with wild-type BlGrpE, the nucleotide exchange factor (NEF) activity of L134H, L134K, L134R, L134D, L134E, L134N, L134Q, L134S, L134G and L134P was reduced by more than 96%. In vitro binding assay revealed that wild-type BlGrpE and the functional variants mainly interacted with the monomer of BlDnaK, but no such interaction was observed for the remaining mutant proteins. BlGrpE and 9 mutant proteins synergistically stimulated the ATPase activity of B. licheniformis DnaK (BlDnaK), whereas the NEF-defective variants had no synergistic stimulation. Comparative analysis of the far-UV CD spectra showed that the α-helical content of the inactive mutant BlGrpEs was reduced significantly with respect to wild-type protein. Moreover, the inactive mutant proteins also exhibited a more sensitivity towards the temperature-induced denaturation. Taken together, these results indicate that Leu134 might play a structural role for the proper function of BlGrpE.
    關聯: Protein Journal 29(5):p.365-372
    Appears in Collections:[ 食品科技系 ] 期刊論文

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