Figure 5. Lid Closure rate is regulated by the NTD-strap.
(A) Continuous Wave (CW) EPR scans of cysteine Free WT (top) and Δstrap zTRAP1 (bottom) labeled with a spin-probe on the NTD-lid (green) in order to observe changes to the lid in the apo and closed states (‘Materials and methods’). In the apo state the lid probe shows signal for both mobile and immobile states, although crystallographic data indicate that even in the mobile state, the majority of the lid is still reasonably well ordered. After addition of AMPPNP, the observed signal shifts indicating a predominantly mobile state of the lid, which corresponds to changes in lid dynamics that accompany NTD rotation and dimerization. Only subtle differences are seen in the mobile:immobile peak ratio upon strap deletion. (B) CW-EPR scans at ∼23°C taken for the cysteine-free WT (red) and Δstrap zTRAP1 (blue) over time after addition of AMPPNP. The percent change in peak height (final vs start) over time is plotted for both the immobile (squares) and mobile (circles) components, showing a clear anti-correlation. The mobile and immobile populations were jointly fit with a single exponential process (‘Materials and methods’) having a rate constant of 0.014 min−1 for WT and 0.075 min−1 for Δstrap, demonstrating a strong coupling between the strap and the NTD-lid.