title: The disassembly and reassembly of mutants of Escherichia coli heat-labile enterotoxin: replacement of proline 93 does not abolish the reassembly-competent and reassembly-incompetent states.
abstract: The carrier moiety of heat-labile enterotoxin of Escherichia coli (EtxB) is formed by the noncovalent association of identical monomeric subunits, which assemble, in vivo and in vitro, into exceptionally stable pentameric complexes. In vitro, acid disassembly followed by neutralization results in reassembly yields of between 20% and 60% depending on the identity of the salts present during the acid denaturation process. Loss of reassembly competence has been attributed to isomerization of the native cis-proline residue at position 93. To characterize this phenomenon further, two mutants of EtxB at proline 93 (P93G and P93A) were generated and purified. The proline variants reveal only minor differences in their biophysical and biochemical properties relative to wild-type protein, but major changes were observed in the kinetics of pentamer disassembly and reassembly. Additionally, a loss of assembly competence was observed following longer term acid treatment, which was even more marked than that of the wild-type protein. We present evidence that the loss of assembly competence of these mutants is best explained by a cis/trans peptidyl isomerization of the unfolded mutant subunits in acid conditions; this limited reassembly competence and the biophysical properties of the native P93 mutant pentamers imply the retention of the native cis conformation in the nonproline peptide bond between residues 92 and 93 in the mutated proteins.
authors.affiliation: Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
authors.forename: C
authors.initials: C
authors.lastname: Cheesman
authors.forename: R B
authors.initials: RB
authors.lastname: Freedman
authors.forename: L W
authors.initials: LW
authors.lastname: Ruddock
mesh_terms: D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics; D011499:Protein Processing, Post-Translational; D020673:Protein Renaturation; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014733:Vibrio / Q000235:genetics
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008024:Ligands; D021122:Protein Subunits; D011994:Recombinant Proteins; D011392:Proline; C027538:heat-labile enterotoxin, E coli; D000409:Alanine; D005998:Glycine