Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese

Markandeswar Panda, Paul M. Horowitz

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM β-ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (Nmutually impliesImutually impliesU) followed by fluorescence at 363 nm, (Nmutually impliesI) by fluorescence at 318 nm, and CD (Imutually impliesU) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5-6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.

Original languageEnglish (US)
Pages (from-to)399-409
Number of pages11
JournalJournal of Protein Chemistry
Issue number5
StatePublished - 2000
Externally publishedYes


  • Active site
  • Molten globule
  • Protein folding
  • Rhodanese
  • Urea denaturation

ASJC Scopus subject areas

  • Biochemistry


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