Table 1.
Summary of binding constants
| CNBD | R348A | mlCNG | |||||
|---|---|---|---|---|---|---|---|
| Ligand | ITC | Stoichiometry | 8-NBD-cAMP fluorescence | Trp fluorescence | 8-NBD-cAMP fluorescence | 8-NBD-cAMP fluorescence | Rb+-flux assay |
| KD (nM) | KD (nM) | KD (nM) | KD (μM) | KD (nM) | K1/2 (nM) | ||
| cAMP | 107±11 (4) | 0.98±0.12 | 67.8±8.7 (7) | 80 (4) | 18.5±4.3 (7) | 81.6±17.5 (6) | 60–100*,† |
| 8-Br-cAMP | 127±11 (3) | 0.95±0.06 | — | — | — | — | |
| 8-CPT-cAMP | 34±25 (3) | 0.91±0.08 | — | — | — | — | |
| 8-NBD-cAMP | 24±1.5 (3) | 0.84±0.10 | 22.0±10.9 (11) | — | 7.3 (1) | 15.9±2.7 (5) | |
| cGMP | 499±69 (3) | 0.97±0.08 | 300.4±15.4 (7) | — | 22.3±5.7 (6) | 320.7±25.5 (6) | 600* |
| 8-Br-cGMP | 236±23 (3) | 1.1±0.06 | — | — | — | — | |
| 8-CPT-cGMP | 2,770±70 (3) | 0.88±0.1 | — | — | — | — | |
CNBD, cyclic nucleotide-binding domain; mlCNG, cyclic nucleotide-gated channel from Mesorhizobium loti; ITC, isothermal titration calorimetry; Trp, tryptophan; *Nimigean et al, 2004; †Clayton et al, 2004