Sulfhydryl modification of E. coli cpn60 leads to loss of its ability to support refolding of rhodanese but not to form a binary complex

Jose A. Mendoza, Paul M. Horowitz

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)589-594
Number of pages6
JournalJournal of Protein Chemistry
Volume11
Issue number6
DOIs
StatePublished - Dec 1992
Externally publishedYes

Keywords

  • Chaperonins
  • chemical modification
  • folding
  • rhodanese

ASJC Scopus subject areas

  • Biochemistry

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