Single-molecule FRET reveals hidden complexity in a protein energy landscape.
Bottom Line: 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.Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function.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.