Cheesman, C. and Ruddock, L.W. and Freedman, R.B. (2004) The refolding and reassembly of Escherichia coli heat-labile enterotoxin B-subunit: Analysis of reassembly-competent and reassembly-incompetent unfolded states. Biochemistry, 43 (6). pp. 1609-1617. ISSN 0006-2960.
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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.
|Additional information:||0006-2960 (Print) Comparative Study Journal Article Research Support, Non-U.S. Gov't|
|Uncontrolled keywords:||Bacterial Toxins/*chemistry/metabolism Buffers Enterotoxins/*chemistry/metabolism Escherichia coli Proteins/*chemistry/metabolism Hydrochloric Acid/chemistry Hydrogen-Ion Concentration Phosphoric Acids/chemistry Potassium Chloride/chemistry Protein Denaturation *Protein Folding *Protein Processing, Post-Translational Protein Renaturation Protein Subunits/*chemistry/metabolism Salts Spectrometry, Fluorescence|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Sue Davies|
|Date Deposited:||10 Sep 2008 14:23|
|Last Modified:||14 Jan 2010 14:25|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/6730 (The current URI for this page, for reference purposes)|
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