TABLE 1.
Comparison of binding constants for cyclic nucleotide binding to the C termini of HCN proteins (HCNCB) derived from FP, SPR, and ITC measurements
|
KD (from FP) ± S.E.a |
EC50 (from SPR) ± S.D. for cAMP | KD (from ITC) ± S.D. for cAMP | ||
|---|---|---|---|---|
| 8-Fluo-cAMP | 8-Fluo-cGMP | |||
| nm | μm | μm | ||
| MBP-HCN1CB | 116 ± 26 (n = 4) | 208 ± 51 (n = 5) | NDb | Not measurable |
| MBP-HCN2CB | 115 ± 18 (n = 4) | 286 ± 34c (n = 5) | ND | 3.06 ± 0.7 (n = 9) |
| MBP-HCN4CB | 111 ± 36 (n = 4) | 414 ± 56d (n = 3) | ND | 0.97 ± 0.7 (n = 4) |
| HCN1CB | 382 ± 87 (n = 4) | 386 ± 76 (n = 3) | 5 ± 1e (n = 5) | Not measurable |
| HCN2CB | 606 ± 219 (n = 3) | 752 ± 236 (n = 3) | 10 ± 4 (n = 3) | 3.6 ± 1.3 (n = 3) |
| HCN4CB | 167 ± 36 (n = 4) | 694 ± 121d (n = 4) | 11 ± 2 (n = 3) | ND |
| HCN1CB ΔC-linker | ND | ND | 5 ± 2e (n = 3) | ND |
| HCN1CB G510S/S515G/S516N | ND | ND | 7.3 ± 1.6e (n = 3) | ND |
a FP experiments were in duplicate or triplicate.
b ND, not determined.
c Mean p < 0.05; values are significantly different from that of 8-Fluo-cAMP (analyzed by paired t test, p value two-tailed, confidence interval 99%, GraphPad Prism version 5.03).
d Mean p < 0.01; values are significantly different from that of 8-Fluo-cAMP (analyzed by paired t test, p value two-tailed, confidence interval 99%, GraphPad Prism version 5.03).
e Protein concentration was 500 nm instead of 100 nm.