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Updated: Jul 10, 2026
Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70
Published on: March 8, 2024
A microplate-based evaluation of complex denaturation pathways: structural stability of Escherichia coli
Jean P Aucamp1, Ruben J Martinez-Torres, Edward G Hibbert
1The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
Abstract:
We have previously developed a rapid microplate-based approach for measuring the denaturation curves by intrinsic tryptophan fluorescence for simple monomeric and two-state unfolding proteins. Here we demonstrate that it can accurately resolve the multiple conformational transitions that occur during the denaturation of a complex multimeric and cofactor associated protein. We have also analyzed the effect of two active-site mutations, D381A and Y440A upon the denaturation pathway of transketolase using intrinsic fluorescence measurements, and we compare the results from classical and microplate-based instrumentation. This work shows that the rapid assay is able to identify changes in the denaturation pathway, due to mutations or removal of cofactors, which affect the stability of the native and intermediate states. This would be of significant benefit for the directed evolution of protein stability, optimizing enzyme stability under biocatalytic process conditions, and also for engineering specific transitions in protein unfolding pathways.
