title: Effects of guanidinium hydrochloride on the structure and immunological properties of Bordetella pertussis fimbriae.
abstract: Denaturation of Bordetella pertussis fimbrial preparations by guanidinium hydrochloride (GdnHCl) has been characterized using static light scattering, c.d., fluorescence and antibody recognition. The susceptibility of Fim2 + 3 (a mixed preparation of two fimbrial types) to GdnHCl was found to be highly dependent on pH; as the pH was increased from pH 7.2 to 10.5, the concentration of GdnHCl required to induce 50% denaturation was decreased. At pH 10.5, Fim2 + 3 was denatured by GdnHCl in a three-step pathway comprising: (1) formation of a pre-denaturational intermediate at less than 1.0 M-GdnHCl; (2) dissociation of the fimbrial polymer into subunits between 2 M- and 3.2 M-GdnHCl; and (3) subunit unfolding between 2.8 M- and 3.6 M-GdnHCl. A similar pathway was also found for the denaturation of the individual fimbrial types, Fim2 and Fim3, except that unfolding of either subunit commenced at a lower GdnHCl concentration (2.2 M) than that found for the mixture of fimbriae, Fim2 + 3. The second step in the denaturation pathway, dissociation into subunits, was partially reversible, but the renaturation and reassociation of fully unfolded subunits to form fimbriae-like structures was not achieved. These findings demonstrate that the GdnHCl denaturation of complex polymeric proteins is unlikely to follow a reversible two-state denaturation pathway, and support the involvement of a chaperone-like protein in the folding and assembly of the fimbriae in vivo. Measurement of the ability of anti-fimbrial monoclonal antibodies to recognize intermediates in the denaturation pathway enabled the identification of two types of epitope which were dependent on different aspects of fimbrial tertiary/quaternary structure.
authors.affiliation: Division of Biotechnology, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
authors.forename: A M
authors.initials: AM
authors.lastname: Pearce
authors.forename: L I
authors.initials: LI
authors.lastname: Irons
authors.forename: A
authors.initials: A
authors.lastname: Robinson
authors.forename: R N
authors.initials: RN
authors.lastname: Seabrook
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology; D001886:Bordetella pertussis / Q000276:immunology / Q000648:ultrastructure*; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D010861:Fimbriae, Bacterial / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D006146:Guanidines; D019791:Guanidine