TABLE 2.
Parameters for wtSkzL and SkzLΔK415 binding to active site fluorescein-labeled Pg/Pm analogs
Dissociation constants for the high affinity (KD,1) and low affinity (KD,2) interactions and corresponding maximum fluorescence changes (ΔFmax,1/Fo) and (ΔFmax,2/Fo) from titrations of [5F]FFR-[Glu]Pg, [5F]FFR-[Lys]Pg, and [5F]FFR-Pm (Probe-labeled protein) with wtSkzL or SkzLΔK415 (Ligand) as indicated. Titrations were performed without polarizers or with vertically polarized excitation and emission at the magic angle (Magic angle) to eliminate anisotropy. Experimental error in the parameters represents ± 2 S.D. Experiments were performed, and the results were analyzed as described under “Experimental Procedures.”
| Probe-labeled protein | Ligand | Magic angle | KD,1 | KD,2 | ΔFmax,1/Fo | ΔFmax,2/Fo |
|---|---|---|---|---|---|---|
| nm | nm | % | % | |||
| [5F]FFR-[Glu]Pg | wtSkzL | No | 10,000 ± 3200 | −18 ± 2 | ||
| [5F]FFR-[Glu]Pg | wtSkzL | Yes | 16,000 ± 9000 | −14 ± 4 | ||
| [5F]FFR-[Glu]Pg | SkzLΔK415 | No | No binding | No binding | ||
| [5F]FFR-[Glu]Pg | SkzLΔK415 | Yes | No binding | No binding | ||
| [5F]FFR-[Lys]Pg | wtSkzL | No | 170 ± 90 | ∼18,000 | −10 ± 2 | −22 ± 10 |
| [5F]FFR-[Lys]Pg | wtSkzL | Yes | 150 ± 40 | −9 ± 1 | ||
| [5F]FFR-[Lys]Pg | SkzLΔK415 | No | 12,000 ± 8000 | −15 ± 5 | ||
| [5F]FFR-[Lys]Pg | SkzLΔK415 | Yes | 2800 ± 1300 | −8 ± 1 | ||
| [5F]FFR-Pm | wtSkzL | No | 160 ± 80 | ∼20,000 | −11 ± 2 | −18 ± 12 |
| [5F]FFR-Pm | wtSkzL | Yes | 140 ± 14 | −14 ± 2 | ||
| [5F]FFR-Pm | SkzLΔK415 | No | 8000 ± 3000 | −18 ± 3 | ||
| [5F]FFR-Pm | SkzLΔK415 | Yes | 4400 ± 1600 | −16 ± 3 |