The refolding and reassembly of Escherichia coli heat-labile enterotoxin B-subunit: analysis of reassembly-competent and reassembly-incompetent unfolded states.
The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is an exceptionally stable protein maintaining its quaternary structure over the pH value range 2.0-11.0. Up to 80% yields of reassembled pentamer can be obtained in vitro from material disassembled for very short incubation periods in KCl-HCl, pH 1.0. However, when the incubation period in acid is extended, the reassembly yield decreases to no more than 20% (Ruddock et al. (1996) J. Biol. Chem. 271 19118-19123). Here we demonstrate that the ion species present in the disassembly conditions strongly influence the reassembly competence of EtxB showing that 60% reassembly yields can be achieved, even after prolonged incubations, by the use of a phosphate buffer for acid disassembly. Using this system, we have fully characterized the disassembly and reassembly behavior of EtxB by electrophoretic, immunochemical, and spectroscopic techniques and compared it with that previously observed. Depending on the denaturation system used, the acid-denatured monomer is either in a predominantly reassembly-competent state (H(3)PO(4) system) or in a predominantly reassembly-incompetent conformation (KCl-HCl system). Interconversion between these two conformations in the denatured state is possible by the addition of salts to the denatured protein. The results are consistent with the previous hypothesis that the conversion between reassembly-competent and -incompetent states corresponds to a cis/trans isomerization of a peptide bond, presumably that to Pro93.
Cheesman
C
C
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Ruddock
L W
LW
Freedman
R B
RB
D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D002021:Buffers; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D006851:Hydrochloric Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010756:Phosphoric Acids / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D020673:Protein Renaturation; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D012492:Salts; D013050:Spectrometry, Fluorescence
D001427:Bacterial Toxins; D002021:Buffers; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D010756:Phosphoric Acids; D021122:Protein Subunits; D012492:Salts; D011189:Potassium Chloride; C027538:heat-labile enterotoxin, E coli; C030242:phosphoric acid; D006851:Hydrochloric Acid