In vivo affinity label of a protein expressed in Escherichia coli

FEBS Letters

Allison, William S.¹  Kiribuchi, Kyoko²  Odaka, Masafumi²  Yoshida, Masasuke² 

[1] University of California at San Diego, La Jolla, CA 92093-0601, USA;Research Laboratory of Resources Utilization, Tokyo Institute af Technology, Nagatsuta 4259, Yokohama 227, Japan

关键词: F1-ATPase;    Affinity label;    CoA;    ATP analogue;    AMEDA;    P1 -(5'-adenosyl)-P2N-(2-mereaptoethyl) diphosphoramidate;    CD;    circular dichroism;    CoA;    Coenzyme A;    DTT;    dithiothreitol;    the β(Y307C);    the mutant β subunit whose tyrosine 307 is replaced by cysteine;    the β(Y307C)r;    the β(Y307C) treated with a reducing sulfhydryl reagent;    Nbf-Cl;    7-chloro-4-nitrobenzofurazan;    MF1;    F1-ATPase from mitochondria;    TF1;    F1-ATPase from a thermophilic Bacillus PS3;    PyDHase;    pyruvate dehydrogenase;    HPLC;    high performance liquid chromatography;    SDS;    sodium dodecylsulfate;   

DOI  :  10.1016/0014-5793(93)80809-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

When Tyr-307 of the β subunit of f₁ -ATPase from a thennophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant β subunit are labeled by Coenzyme A at Cys-307 through a disulfide bond which is cleavable by reducing treatment. The mutant β subunit can be reconstituted into the α₃β₃, complex of which ATPase activity is stimulated two-fold by reducing treatment either prior or after reconstitution. Since Tyr-307 has been supposed to be located at one of subdomains which form the ATP binding site of the β subunit, Coenzyme A binds to the mutant β subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys-307.

【 授权许可】

Unknown