title: Equilibrium folding of pro-HlyA from Escherichia coli reveals a stable calcium ion dependent folding intermediate.
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 Ca(2+) 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 Ca(2+) is essential for stability as well as correct folding of the whole protein. In the absence of Ca(2+), pro-HlyA adopts a non-native conformation. Such molecules could however be rescued by Ca(2+) addition, indicating that these are not dead-end species and that Ca(2+) 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 6-7M urea, relates to Ca(2+) 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 Ca(2+) induced folding is not restricted to the RTX domain. Taken together, Ca(2+) binding to the pro-HlyA RTX domain is required to drive the folding of the entire protein to its native conformation.
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr 1, 40225 Düsseldorf, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Thomas
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr 1, 40225 Düsseldorf, Germany.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Bakkes
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr 1, 40225 Düsseldorf, Germany.
authors.forename: Sander H J
authors.initials: SH
authors.lastname: Smits
authors.affiliation: Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstr 1, 40225 Düsseldorf, Germany. Electronic address: lutz.schmitt@hhu.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Schmitt
mesh_terms: D058947:Bacterial Secretion Systems / Q000502:physiology; D002118:Calcium / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry; D014508:Urea / Q000737:chemistry
chemical_list: D058947:Bacterial Secretion Systems; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; D011498:Protein Precursors; D014364:Tryptophan; D014508:Urea; D002118:Calcium
keywords: Calcium; Folding mechanics; HlyA; Protein folding; RTX; Tryptophan fluorescence