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Single-molecule FRET reveals hidden complexity in a protein energy landscape.

Tsytlonok M, Ibrahim SM, Rowling PJ, Xu W, Ruedas-Rama MJ, Orte A, Klenerman D, Itzhaki LS - Structure (2015)

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.

View Article: PubMed Central - PubMed

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.

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Effects of Urea on the FRET Z Parameter Histograms of the Labeled Wild-Type D34 and Variants(A) FRET Z parameter histograms are shown in the absence of urea (gray) and in the presence of 7 M urea (blue). The full urea titrations are shown in Figure S6.(B) The number of molecules as measured by smFRET using single (blue) laser excitation (1c) and TCCD using dual laser (blue and red) excitation (2c) are plotted as a function of urea concentration.
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fig2: Effects of Urea on the FRET Z Parameter Histograms of the Labeled Wild-Type D34 and Variants(A) FRET Z parameter histograms are shown in the absence of urea (gray) and in the presence of 7 M urea (blue). The full urea titrations are shown in Figure S6.(B) The number of molecules as measured by smFRET using single (blue) laser excitation (1c) and TCCD using dual laser (blue and red) excitation (2c) are plotted as a function of urea concentration.

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).


Single-molecule FRET reveals hidden complexity in a protein energy landscape.

Tsytlonok M, Ibrahim SM, Rowling PJ, Xu W, Ruedas-Rama MJ, Orte A, Klenerman D, Itzhaki LS - Structure (2015)

Effects of Urea on the FRET Z Parameter Histograms of the Labeled Wild-Type D34 and Variants(A) FRET Z parameter histograms are shown in the absence of urea (gray) and in the presence of 7 M urea (blue). The full urea titrations are shown in Figure S6.(B) The number of molecules as measured by smFRET using single (blue) laser excitation (1c) and TCCD using dual laser (blue and red) excitation (2c) are plotted as a function of urea concentration.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291146&req=5

fig2: Effects of Urea on the FRET Z Parameter Histograms of the Labeled Wild-Type D34 and Variants(A) FRET Z parameter histograms are shown in the absence of urea (gray) and in the presence of 7 M urea (blue). The full urea titrations are shown in Figure S6.(B) The number of molecules as measured by smFRET using single (blue) laser excitation (1c) and TCCD using dual laser (blue and red) excitation (2c) are plotted as a function of urea concentration.
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).

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus