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 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.
Highlights
The serpin superfamily, with members in every phylogenetic kingdom, underwent a marked expansion in diversity following the plant-animal split[1,2,3]
As the interaction between two defined partners is generally determined by multiple specific subsite interactions within the binding cleft[35], this consensus reactive centre loop (RCL) may not fully capture the potential inhibitory activity of the conserpin scaffold against a single target
Serpins are present in every taxonomic phylum, including prokaryotes that live at extremes of temperature
Summary
The serpin superfamily, with members in every phylogenetic kingdom, underwent a marked expansion in diversity following the plant-animal split[1,2,3]. Mutations have systematically been identified that are compatible with function and enhance stability for a range of proteins with include DNA binding proteins[29], antibodies[30,31], leucine rich repeat proteins[32], and enzymes[33] By extending this approach to the whole protein rather than selected residues, conserpin, an entirely artificial serpin, has recently been engineered[34] with a sequence that reflects the most frequently observed residue at each site in an alignment of eukaroytic serpins[1]. This protein was found to adopt the canonical native, metastable conformation and showed a remarkable stability to inactivation by heat, an atypical fully reversible unfolding pathway, and an absence of polymer formation. The data reflect a molecule with a highly efficient, concerted folding mechanism that minimises the accumulation of an aggregation-prone intermediate species as a result of a pronounced kinetic stability
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