Fig. 5: The influence of GroEL and GroEL/ES/ATP on the folding landscape of DM-MBP.

a–d 2D FRET efficiency vs donor lifetime (τ_D(a)) histograms (E-τ plot) of the NTD during refolding in 0.1 M GuHCl. The refolding of the NTD is shown for a spontaneous refolding, b in presence of 3 µM GroEL, and (c–d) upon the addition of 3 µM GroEL, 6 µM GroES and 2 mM ATP. Only the initial 10 minutes of all the three measurements are shown for (a–c) and between 10 and 20 min for (d). Above and to the right, 1-D projections are shown. The static-FRET line (black) and a dynamic-FRET line (red) are plotted for comparison. The end points for the dynamic FRET line were determined by fitting the subensemble donor fluorescence lifetimes to a biexponential. The schematic below each panel illustrates the experiment and the conformation of GroEL during the folding cycle. e A schematic of the protein-folding funnel describing the refolding of WT-MBP, DM-MBP and the role of chaperones on the folding energy landscape. In the case of WT-MBP (grey), the NTD is guided by hydrophobic interactions and folds first followed by CTD folding. In the case of DM-MBP (brown), NTD folding is delayed by the high configurational entropy generated by the loss of binding energy due to less hydrophobic mutations. As soon as the NTD finds its folding competent conformation in DM-MBP, the CTD folds along the folding funnel as for WT-MBP. Chaperones shape the protein folding funnel (beige) by restricting the conformational space available to the substrate, thereby guiding the protein towards the correct conformation and thereby accelerating the refolding rate.