Mendoza J A, Horowitz P M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
J Protein Chem. 1992 Dec;11(6):589-94. doi: 10.1007/BF01024958.
Differential chemical modification of E. coli chaperonin 60 (cpn60) was achieved by using one of several sulfhydryl-directed reagents. For native cpn60, the three cysteines were accessible for reaction with N-ethylmaleimide (NEM), while only two of them are accessible to the larger reagent 4,4'-dipyridyl disulfide (4-PDS). However, no sulfhydryl groups were modified when the even larger reagents 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or 2-(4'-(iodoacetamido)anilino) naphthalene-6-sulfonic acid (IAANS), were employed, unless the chaperonin was unfolded. The cpn60 that had been covalently modified with NEM or IAANS, was not able to support the chaperonin-assisted refolding of the mitochondrial enzyme rhodanese, which also requires cpn10 and ATP hydrolysis. However, both modified forms of cpn60 were able to form binary complexes with rhodanese, as demonstrated by their ability to arrest the spontaneous refolding of the enzyme. That is, chemical modification with these sulfhydryl-directed reagents produced a species that was not prevented from interaction with partially folded rhodanese, but that was prevented from supporting a subsequent step(s) during the chaperonin-assisted refolding process.
通过使用几种巯基导向试剂中的一种，实现了对大肠杆菌伴侣蛋白60（cpn60）的差异化学修饰。对于天然cpn60，三个半胱氨酸可与N-乙基马来酰亚胺（NEM）反应，而对于较大的试剂4,4'-二吡啶二硫化物（4-PDS），只有其中两个半胱氨酸可与之反应。然而，当使用更大的试剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB）或2-（4'-（碘乙酰胺基）苯胺基）萘-6-磺酸（IAANS）时，除非伴侣蛋白展开，否则没有巯基被修饰。用NEM或IAANS共价修饰的cpn60不能支持伴侣蛋白辅助的线粒体酶硫氰酸酶的重折叠，硫氰酸酶的重折叠也需要cpn10和ATP水解。然而，cpn60的两种修饰形式都能够与硫氰酸酶形成二元复合物，这可通过它们阻止该酶自发重折叠的能力来证明。也就是说，用这些巯基导向试剂进行化学修饰产生了一种物质，它不会被阻止与部分折叠的硫氰酸酶相互作用，但会被阻止在伴侣蛋白辅助的重折叠过程中支持后续步骤。
