Probing the folding pathway of a consensus serpin using single tryptophan mutants

Li Yang, James A. Irving, Weiwen Dai, Marie-Isabel Aguilar, Stephen P. Bottomley

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7 Citations (Scopus)


Conserpin is an engineered protein that represents the consensus of a sequence alignment of eukaryotic serpins: protease inhibitors typified by a metastable native state and a structurally well-conserved scaffold. Previously, this protein has been found to adopt a native inhibitory conformation, possess an atypical reversible folding pathway and exhibit pronounced resistance to inactivation. Here we have designed a version of conserpin, cAT, with the inhibitory specificity of α1-antitrypsin, and generated single-tryptophan variants to probe its folding pathway in more detail. cAT exhibited similar thermal stability to the parental protein, an inactivation associated with oligomerisation rather a transition to the latent conformation, and a native state with pronounced kinetic stability. The tryptophan variants reveal the unfolding intermediate ensemble to consist of an intact helix H, a distorted helix F and 'breach' region structurally similar to that of a mesophilic serpin intermediate. A combination of intrinsic fluorescence, circular dichroism, and analytical gel filtration provide insight into a highly cooperative folding pathway with concerted changes in secondary and tertiary structure, which minimises the accumulation of two directly-observed aggregation-prone intermediate species. This functional conserpin variant represents a basis for further studies of the relationship between structure and stability in the serpin superfamily.

Original languageEnglish
Article number2121
Number of pages15
JournalScientific Reports
Issue number1
Publication statusPublished - 1 Feb 2018


  • biophysical chemistry
  • molecular evolution
  • protein design
  • protein folding
  • synthetic biology

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