Mutation in the interdomain tether influences the stability and refolding of the enzyme rhodanese

Guang Xiang Luo, Su Hua, Paul M. Horowitz

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Rhodanese is a single polypeptide chain of 293 amino acids that is folded into two globular domains of nearly equal size that are connected by a 16 amino acid tether. Two amino acids, Val-Asp (VD), were inserted into the interdomain tether through site-directed mutagenesis to produce the new interdomain sequence, E145PSRPEPAIFKAVDTLNR. The purified mutant protein, when unperturbed, was virtually indistiguishable in all properties tested and gave a specific activity that was at least 90% of the WT. However, the tether mutant was considerably less stable to perturbation compared with the WT enzyme. The interdomain hydrophobic surfaces in the mutant were more easily exposed, and the formation of intermediate folding states was facilitated. The rate of unassisted refolding was slightly less for the mutant, and the yield of active enzyme was somewhat reduced. The mutation introduced a new V8 proteinase cleavage site, but this site was not accessible in the native mutant which was as resistant to proteolysis as the WT enzyme. However, perturbation with low concentrations of urea that could form folding intermediate(s), allowed facile cleavage of the mutant to give fragments that appeared to represent the individual domains. In addition, the perturbed mutant could be proteolyzed close to one end of the polypeptide, a position that is far from the site of mutation, and which was not readily cleaved in the WT enzyme or the native form of the mutant. These results indicate that mutation in the interdomain tether can have dramatic effects on the stability and conformational transitions of rhodanese.

Original languageEnglish (US)
Pages (from-to)165-171
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1252
Issue number1
DOIs
StatePublished - Sep 27 1995
Externally publishedYes

Keywords

  • Domain
  • Protein folding
  • Rhodanese
  • Site-directed mutation
  • Tether

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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