(a) HAP-TAMRA in solution for a matched pair of samples with 8 µM capsid (colored lines) and without (black lines). In the presence of capsid, the absorbance spectrum shifts towards one that resembles a π-stacked TAMRA dimer (Adachi et al., 2014). The change is not as dramatic as in Figure 2c, due to the presence of free dye in sample that have not been purified by SEC. (b) The change in absorbance at 520 nm can be used to observe kinetics of binding different HAP-TAMRA concentrations to 8 µM capsid, showing the time dependence of forming the samples in panel a. The kinetic traces are averages of three independent experiments. The dashed lines fit to a two-phase exponential association. The half-life for binding is between 5 and 10 min. (c) Further normalization of the kinetic curves by their maximum value gives a visual expression of the similarity of the half-lives, and reiterates that the kinetics appear to be similar across input concentrations (d) The time course in b was monitored for 350 min. Shown are the endpoint values of the change in 520 nm absorbance. The result is comparable to the one in Figure 2d, where the bound probe is isolated from the free probe. (e) Assuming the maximum amount of HAP-TAMRA binds to the protein in each condition, we can calculate the concentration of HAP-TAMRA bound. Plotting extinction coefficient for the ΔA520nm signal is constant across the titration, with a value of 11196 M−1 ± 4%. (f) A demonstration that fluorescence is also quenched upon binding using 40 µM HAP-TAMRA, 8 µM Cp dimer. In the presence of capsid, we estimate that 75% of the HAP-TAMRA was unbound, accounting for most of the residual fluorescence.