Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate |
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Authors: | Sabrina Thomas Patrick J. BakkesSander H.J. Smits Lutz Schmitt |
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Affiliation: | Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr 1, 40225 Düsseldorf, Germany |
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Abstract: | HlyA from Escherichia coli is a member of the repeats in toxin (RTX) protein family, produced by a wide range of Gram-negative bacteria and secreted by a dedicated Type 1 Secretion System (T1SS). RTX proteins are thought to be secreted in an unfolded conformation and to fold upon secretion by Ca2 + binding. However, the exact mechanism of secretion, ion binding and folding to the correct native state remains largely unknown. In this study we provide an easy protocol for high-level pro-HlyA purification from E. coli. Equilibrium folding studies, using intrinsic tryptophan fluorescence, revealed the well-known fact that Ca2 + is essential for stability as well as correct folding of the whole protein. In the absence of Ca2 +, pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca2 + addition, indicating that these are not dead-end species and that Ca2 + drives pro-HlyA folding. More importantly, pro-HlyA unfolded via a two-state mechanism, whereas folding was a three-state process. The latter is indicative of the presence of a stable folding intermediate. Analysis of deletion and Trp mutants revealed that the first folding transition, at 6–7 M urea, relates to Ca2 + dependent structural changes at the extreme C-terminus of pro-HlyA, sensed exclusively by Trp914. Since all Trp residues of HlyA are located outside the RTX domain, our results demonstrate that Ca2 + induced folding is not restricted to the RTX domain. Taken together, Ca2 + binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation. |
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Keywords: | AP, alkaline protease CBB, Coomassie Brilliant Blue SEC, size exclusion chromatography RTX, repeats in toxin T1SS, type 1 secretion system |
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