Probing the folding pathway of a consensus serpin using single tryptophan mutants
- Author(s)
- Yang, L; Irving, JA; Dai, W; Aguilar, MI; Bottomley, SP;
- Details
- Publication Year 2018-02-01,Volume 8,Issue #1,Page 2121
- Journal Title
- Scientific Reports
- Publication Type
- Journal Article
- Abstract
- 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 alpha1-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.
- Publisher
- Springer Nature
- PubMed ID
- 29391487
- Publisher's Version
- https://doi.org/10.1038/s41598-018-19567-9
- Open Access at Publisher's Site
https://doi.org/10.1038/s41598-018-19567-9- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2018-02-28 08:59:28
Last Modified: 2018-02-28 09:02:57