Figure 4.

Time-dependent ensemble fluorometry. (a) HyCbl binding quenches Cy3 (see also Supporting Information Figure 2). (b) The single-exponential decay of the Cy3 fluorescence reports on the sum of the apparent binding (^appk_bind) and dissociation (^appk_diss) rate constants associated with the interaction between HyCbl and the env8HyCbl riboswitch. Elevated concentrations of ligand increase the rate constant for establishing equilibrium. (c) A linear-fit of the equilibration rate constant as a function of [HyCbl] is used to measure the apparent ligand binding (^appk_bind) and dissociation (^appk_diss) rate constants.