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. Author manuscript; available in PMC: 2015 Oct 17.
Published in final edited form as: J Am Chem Soc. 2014 Nov 19;136(48):16832–16843. doi: 10.1021/ja5076184

Figure 7.

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 (kundock).