The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure. Study holds ProTherm entries: 4574 Extra Details: additive : Na2EDTA(0.1 mM), secondary structure; hydrophobic surface; two-state model;,association reactions
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points||3|
|Proteins||Trp operon repressor ; Trp operon repressor|
|Assays/Quantities/Protocols||Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence AQQSPYSAAMAEQRHQEWLRFVDLLKNAYQNDLHLPLLNLMLTPDEREALGTRVRIVEELLRGEMSQRELKNELGAGIATITRGSNSLKAAPVELRQWLEEVLLKSD|