title: A kinetic model of intermediate formation during assembly of cholera toxin B-subunit pentamers.
abstract: Cholera toxin is the most important virulence factor produced by Vibrio cholerae. The pentameric B-subunit of the toxin can bind to GM1-ganglioside receptors, leading to toxin entry into mammalian cells. Here, the in vitro disassembly and reassembly of CtxB(5) (the B subunit pentamer of cholera toxin) is investigated. When CtxB(5) was acidified at pH 1.0 and then neutralized, the B-subunits disassembled and could no longer migrate as SDS-stable pentamers on polyacrylamide gels or be captured by GM1. However, continued incubation at neutral pH resulted in the B-subunits regaining the capacity to be detected by GM1 enzyme-linked immunosorbent assay (t(12) approximately 8 min) and to migrate as SDS-stable pentamers (t(12) approximately 15 min). Time-dependent changes in Trp fluorescence intensity during B-subunit reassembly occurred with a half-time of approximately 8 min, similar to that detected by GM1 enzyme-linked immunosorbent assay, suggesting that both methods monitor earlier events than B-pentamer formation alone. Based on the Trp fluorescence intensity measurements, a kinetic model of the pathway of CtxB(5) reassembly was generated that depended on trans to cis isomerization of Pro-93 to give an interface capable of subunit-subunit interaction. The model suggests formation of intermediates in the reaction, and these were successfully detected by glutaraldehyde cross-linking.
authors.affiliation: Department of Pathology, School of Medical Sciences University of Bristol, Bristol, United Kingdom.
authors.forename: Claire
authors.initials: C
authors.lastname: Lesieur
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cliff
authors.forename: Rachel
authors.initials: R
authors.lastname: Carter
authors.forename: Roger F L
authors.initials: RF
authors.lastname: James
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Clarke
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Hirst
mesh_terms: D002772:Cholera Toxin / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014733:Vibrio / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D012967:Sodium Dodecyl Sulfate; D005677:G(M1) Ganglioside; D014364:Tryptophan; D002772:Cholera Toxin; D011392:Proline; D005976:Glutaral