title: A microplate-based evaluation of complex denaturation pathways: structural stability of Escherichia coli transketolase.
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.
authors.affiliation: The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK.
authors.forename: Jean P
authors.initials: JP
authors.lastname: Aucamp
authors.forename: Ruben J
authors.initials: RJ
authors.lastname: Martinez-Torres
authors.forename: Edward G
authors.initials: EG
authors.lastname: Hibbert
authors.forename: Paul A
authors.initials: PA
authors.lastname: Dalby
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D040081:Protein Array Analysis / Q000379:methods*; D011489:Protein Denaturation; D015398:Signal Transduction / Q000502:physiology*; D014174:Transketolase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D014174:Transketolase