Fig. 2: A conformational search during DM-MBP refolding is the cause for the entropic barrier governing unfolding/refolding hysteresis.
a Equilibrium refolding of the DM-MBP NTD, CTD and N-C interface displayed as waterfall plots of FRET Efficiency versus GuHCl concentration. The burst-averaged FRET efficiencies were compared with the FRET efficiencies obtained from the two donor lifetime components determined from a fit to the photons detected from all bursts (white squares) (Table S4). A grey line is shown at 0.1 M GuHCl concentration where the NTD and N-C interface constructs exhibit a mostly refolded conformation, whereas a substantial fraction of molecules for the CTD construct are still in the intermediate population. A white line separates the 0.9 M GuHCl measurement from the higher denaturant concentrations. b 2D-plots of FRET efficiency vs donor lifetime in the presence of an acceptor (τD(a)) (E-τ plot) is shown for the DM-MBP NTD, CTD and N-C experiments at all GuHCl concentrations displayed in panel A. The static-FRET line and a dynamic-FRET line for the measurement at 0.9 M GuHCl are shown in black and red, respectively (See Supplementary Note 2 for details). The color legend for occurrences for both panels A and B is shown in between the two panels. c A waterfall plot of FRET efficiency versus GuHCl concentration is shown for equilibrium refolding measurements of the CTD of WT-MBP. A white line separates the measurements below and above 0.9 M GuHCl concentration, a concentration below which the dynamic intermediate population is significantly populated during DM-MBP refolding. Contrary to the refolding traces of the NTD, CTD, and the N-C interface for DM-MBP, an intermediate population is not clearly visible for WT-MBP refolding. d The influence of the chemical chaperone trimethylamine-N-oxide (TMAO). Upper panel: A smFRET histogram for DM-MBP NTD refolding in 0.2 M GuHCl, where a significant fraction of the intermediate population (yellow) is observed compared to the unfolded (green) and refolded states (orange). Lower panel: A smFRET histogram for NTD refolding in 0.2 M GuHCl where 500 mM TMAO has been added. The unfolded state is no longer visible and the intermediate population has diminished (yellow vs orange).
