Table 1.
Rate constants for a variety of protein–ligand interactions
| Ligand | Buffer | T (°C) | KdPapp (nM) | Competitor | k−1P (s−1) | kθD (M−1s−1) |
|---|---|---|---|---|---|---|
| r(GGAA)10[A488] | BB25 | 25 | *78 ± 12 | r(GGAA)10 | *,†n.d. | *,†n.d. |
| 4 to 25 | n.d. | r(GGAA)10 | *5.2 ± 0.5 (×10−4) | *79 ± 6.0 | ||
| r(G3A2)4[F] | BB10 | 25 | ‡2.3 ± 0.35 | ds-d(N)50 | ||1.4 ± 1.1 (×10−3) | ||91 ± 31 |
| 4 to 25 | n.d. | r(G3A2)4 | 8.3 ± 1.6 (×10−4) | 30 ± 13 | ||
| ds-d(N)60 | ||4.5 ± 0.15 (×10−4) | ||100 ± 19 | ||||
| BB25 | 25 | 4.4 ± 0.34 | r(G3A2)4 | 1.7 ± 0.60 (×10−3) | 660 ± 130 | |
| 4 to 25 | n.d. | r(G3A2)4 | 5.6 ± 0.50 (×10−4) | 47 ± 17 | ||
| §r(G3A2)4 | C1 | ¶4.7 × 10−4 | ¶73 | ||||
| BB100 | 25 | ‡1.4 ± 0.15 | – | – | – | |
| BB200 | 25 | 6.0 ± 0.93 | – | – | – | |
| r(G3A2)4[A488] | BB25 | 25 | 8.2 ± 0.69 | – | – | – |
| 4 to 25 | n.d. | r(G3A2)4 | 8.8 ± 0.32 (×10−4) | 59 ± 20 | ||
| ds-[F]d(N)60 | BB10 | 25 | 5.1 ± 0.60 | r(G3A2)4 | 2.4 ± 0.25 (×10−3) | 170 ± 41 |
| ds-d(N)60 | ¶9.1 × 10−5 | ¶260 | ||||
| 4 to 25 | n.d. | r(G3A2)4 | 1.2 ± 0.062 (×10−3) | 67 ± 8.8 | ||
| ds-d(N)60 | 5.3 ± 2.3 (×10−4) | 150 ± 21 | ||||
| BB25 | 25 | 82 ± 13 | – | – | – | |
| BB100 | 25 | 170 ± 15 | – | – | – | |
| BB200 | 25 | #n/a | – | – | – | |
| ds-d(N)50[F] | BB10 | 25 | 5.0 ± 0.46 | r(G3A2)4 | 2.5 ± 0.11 (×10−3) | 170 ± 8.2 |
| ds-d(N)50 | 7.6 ± 0.75 (×10−4) | 210 ± 91 | ||||
| 4 to 25 | n.d. | ds-d(N)50 | ¶2.8 × 10−4 | ¶340 | ||
| BB25 | 25 | 390 ± 31 | – | – | – |
*Experiments used PRC25m (somatic AEBP2 isoform), not PRC25me (embryonic AEBP2 isoform).
†Dissociation completed during initiation-measurement delay (~90 s; λ ≥ 3.3 × 10−2 s−1).
‡Experiment used [Ligand] ≥ 2× KdPapp; it’s possible that Kd < Kdapp.
§Total polynucleotide concentration was kept constant by serially diluting competitor in a carrier nucleic acid; C1 = r(A)20.
¶Value from single experiment.
#Binding too weak to obtain Kdapp (> 1 µM).
||Weak competitor—manual baseline (from binding curve) used for regression calculations.
Fluorescence polarization-based methodology (Fig. 1 and Materials and Methods) was used to determine the apparent equilibrium dissociation constants (KdPapp) (in the absence of competitor), intrinsic dissociation rate constants (k−1P), and direct transfer rate constants (kθD). Values indicate mean ± SD for at least three independent experiments. Kdapp are from regression with a standard (non-quadratic, non-Hill) binding equation (SI Appendix, Eq. S2). Numerical subscript of buffers refers to their variable concentration of salt, and specific buffer definitions can be found in Materials and Methods. Additional nomenclature definition is provided in SI Appendix, Table S1, and polynucleotide sequences are defined in SI Appendix, Table S2.
n.d. = not determined; n/a = not applicable.