Figure 3.

Rad50 D-loop mutations form less ‘closed’ state. (A and B) Distance distributions derived from exponential series method analysis of the LRET acceptor intensity decays. Distributions of the apo and ATP-bound states are shown for distances between Tb³⁺ (donor) and Bodipy FL (Bo; acceptor) labeled wild type (green), L828F (orange), D829N (blue), L828A (purple), and D829A (light blue) Rad50 NBDs in full-length MR (A) or truncated MR^NBD (B) complexes. Vertical dashed lines represent the average distance between donor and acceptor (from three measurements; see Table 1) of the closed and ‘partially open’ conformations for ATP-bound wild type MR. The schematic on top highlights that the closed (∼38 Å) and ‘partially open’ (∼48–55 Å) conformations are within the sensitive range of the LRET probes, while NBDs farther apart than ∼60 Å (i.e. open conformations) are invisible. (C) LRET time-based data where donor–sensitized acceptor intensity was measured after the addition of ATP to the indicated concentrations of MR^NBD complexes. The intensities for mutant curves are scaled by a factor of 1.5 for graphical purposes. Inset compares the equilibration rates relative to wild type MR.