Effects of guanidinium hydrochloride on the structure and immunological properties of Bordetella pertussis fimbriae.
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.
Pearce
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AM
Division of Biotechnology, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.K.
Irons
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LI
Robinson
A
A
Seabrook
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RN
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
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D006146:Guanidines; D019791:Guanidine