Figure 7.

Surface immobilized KL docking kinetics in the presence of hydroxocobalamin (HyCbl). (a) Representative fluorescence trajectory from a surface immobilized molecule in the presence of 2.5 nM HyCbl. For a construct in the undocked conformation, ligand binding (arrow) significantly diminishes the fluorescence intensity, which returns back to normal after ligand release. (b) When the ligand is not bound, the docking/undocking kinetics of the regulatory KL are experimentally indistinguishable from those in the absence of HyCbl (see also Figure 6b). (c) When the ligand is bound, a maximum-likelihood 2-state model (black line) is used to quantify the dwell times associated with fluctuations in the acceptor fluorescence resulting from KL docking and undocking. A comparison between the two KL docking equilibria (b vs c) reveals that the effect of HyCbl binding significantly decreases the undocking rate constant (k_undock).