Moe Luke A, McMartin Lea A, Fox Brian G
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706-1544, USA
Biochemistry. 2006 May 2;45(17):5478-85. doi: 10.1021/bi0601611.
A fluorophore-labeled form of the T4moD, the catalytic effector protein of the toluene 4-monooxygenase complex, was prepared by engineering the N-terminal region to contain a tetraCys motif and treatment with biarsenical fluorescein. Fluorescence anisotropy was used to study the protein-protein interactions among various combinations of the four components of the complex. Binding interactions were detected between T4moD and the hydroxylase component T4moH [K(D) value of 83 nM for interaction with the alphabetagamma protomer] and between T4moD and the Rieske [2Fe-2S] ferredoxin component T4moC (K(D) value of 78 nM). No binding interactions were detected between T4moD and the NADH oxidoreductase component T4moF, but T4moF was able to disrupt binding between T4moC and T4moD. The detected binding interactions suggest an intermediary electron transfer complex between T4moC and T4moD that excludes T4moF. The results indicate that specialization of effector protein function may include specific protein-protein interactions with [2Fe-2S] domains as well as the hydroxylase component.
通过对甲苯4-单加氧酶复合物的催化效应蛋白T4moD的N端区域进行工程改造，使其包含一个四半胱氨酸基序，并使用双砷荧光素进行处理，制备了一种荧光团标记形式的T4moD。荧光各向异性用于研究该复合物四个组分的各种组合之间的蛋白质-蛋白质相互作用。在T4moD与羟化酶组分T4moH之间检测到结合相互作用（与字母γ原聚体相互作用的K(D)值为83 nM），以及在T4moD与 Rieske [2Fe-2S]铁氧还蛋白组分T4moC之间检测到结合相互作用（K(D)值为78 nM）。在T4moD与NADH氧化还原酶组分T4moF之间未检测到结合相互作用，但T4moF能够破坏T4moC与T4moD之间的结合。检测到的结合相互作用表明在T4moC与T4moD之间存在一个排除T4moF的中间电子传递复合物。结果表明效应蛋白功能的特化可能包括与[2Fe-2S]结构域以及羟化酶组分的特定蛋白质-蛋白质相互作用。
