Cheesman, C. and Freedman, R.B. and Ruddock, L.W. (2004) 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. Biochemistry, 43 (6). pp. 1618-1625. ISSN 0006-2960.
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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.
|Additional information:||0006-2960 (Print) Comparative Study Journal Article Research Support, Non-U.S. Gov't|
|Uncontrolled keywords:||Alanine/genetics Amino Acid Substitution/*genetics Bacterial Toxins/*chemistry/genetics/metabolism Enterotoxins/*chemistry/genetics/metabolism Escherichia coli Proteins/*chemistry/genetics/metabolism Glycine/genetics Hydrogen-Ion Concentration Kinetics Ligands *Mutagenesis, Site-Directed Proline/*genetics Protein Binding/genetics Protein Conformation Protein Denaturation/genetics *Protein Processing, Post-Translational Protein Renaturation Protein Subunits/chemistry/genetics/metabolism Recombinant Proteins/biosynthesis/chemistry/metabolism Vibrio/genetics|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences > Protein Science Group|
|Depositing User:||Sue Davies|
|Date Deposited:||10 Sep 2008 14:20|
|Last Modified:||14 Jan 2010 14:25|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/6729 (The current URI for this page, for reference purposes)|
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