Single-molecule FRET reveals hidden complexity in a protein energy landscape.
Bottom Line: We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR.Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane.Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.
Affiliation: MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.Show MeSH
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Mentions: Single-molecule urea-induced equilibrium unfolding experiments were performed next (see Figure 2 for low and high urea concentrations and Figure S3 for the complete set of urea concentrations). As discussed earlier, the histograms do not follow simple Gaussian distribution, and, therefore, the Z parameter histogram was applied. For ANK3-5, the FRET Z parameter histogram, which shows a peak at a Z value of ∼1 at 0 M urea, becomes broader at urea concentrations of 1.5, 2, and 2.5 M. These data can be fitted to Gaussian with two peaks at Z values around 1 and −1. Above 3 M urea, only the peak with the Z value of −1 is observed. These results are consistent with unfolding of the protein leading to separation of the dye pairs. The transition between high and low Z values is similar to the unfolding transition measured by dye fluorescence in bulk experiments (Figure S1D; Table S2) and is also in agreement with the lifetime measurements as a function of urea (Figure S2). The results are consistent with a cooperative mode of unfolding for the N-terminal subdomain of D34, as observed previously in ensemble experiments (Werbeck and Itzhaki, 2007).
Affiliation: MRC Cancer Cell Unit, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK; Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.