FIGURE 5.
Binding of wtSkzL and SkzLΔK415 to [5F]FFR-[Glu]Pg, [5F]FFR-[Lys]Pg, and [5F]FFR-Pm. A and B, titrations of the fractional change in fluorescence (ΔF/Fo) of 50 nm [5F]FFR-[Glu]Pg as a function of total wtSkzL concentration ([SkzL]o) in the absence (●) and presence (○) of 10 mm 6-AHA and as a function of total SkzLΔK415 concentration ([SkzLΔK415]o) in the absence (▴) and presence (Δ) of 10 mm 6-AHA. C and D, titrations as in A and B of 50 nm [5F]FFR-[Lys]Pg as a function of total wtSkzL concentration in the absence (●) and presence (○) of 10 mm 6-AHA and as a function of total SkzLΔK415 concentration in the absence (▴) and presence (Δ) of 10 mm 6-AHA. E and F, titrations as in A and B of 50 nm [5F]FFR-Pm as a function of total wtSkzL concentration in the absence (●) and presence (○) of 10 mm 6-AHA, and as a function of total SkzLΔK415 concentration in the absence (▴) and presence (Δ) of 10 mm 6-AHA. Solid lines represent the least squares fits of the data by Equation 1 (A, B, D, and F) or Equation 2 (C and E) with the parameters listed in Table 2. The dashed line represents zero. Titrations were performed without polarizers (A, C and E), or with vertically polarized excitation and emission at the magic angle (B, D, and F). Titrations were performed and analyzed as described under “Experimental Procedures.”