In vivo affinity label of a protein expressed in Escherichia coli

[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.

[学科分类] 生物化学/生物物理

[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