Table 2. FRET constructs derived from the cNMP-BD of cGKI.
Shown are the relative changes in CFP/YFP emission ratio of the constructs derived from the cNMP-BD of cGK determined in vitro as described in the Materials and methods section. The borders of the constructs containing either cNMP-BD A+B or cNMP-BD A only (see Figure 1) are stated relative to the border of the cNMP-BD (left column) of bovine cGKI (GenBank® accession number X16086.1 [44]). The first and the last amino acid (aa) relative to the cGKI holoenzyme are given in parentheses. Results are means±S.E.M. three or more independent experiments. n.d., not determined because no ratio change was detectable.
| Indicator construct | N-terminal elongation (first aa) | C-terminal elongation (last aa) | Change in CFP/YFP emission ratio (%) | EC50 (μM) | |
|---|---|---|---|---|---|
| cNMP-BD A+B (Gln79–Tyr336) | +45 (Gln34) | 0 (Tyr336) | 29±3 | 1.47±0.44 | |
| +9 (Tyr345) | 27±2 | 0.64±0.19 | |||
| +35 (Gln44) | 0 (Tyr336) | 42±2 | 1.25±0.20 | ||
| +9 (Tyr345) | 45±2 | 0.23±0.04 | |||
| +23 (Pro56) | 0 (Tyr336) | 56±2 | 1.11±0.13 | ||
| +9 (Tyr345) | 55±3 | 0.27±0.02 | |||
| +12 (Glu67) | 0 (Tyr336) | 66±2 | 1.57±0.04 | ||
| +9 (Tyr345) | 55±3 | 0.50±0.06 | |||
| +5 (Phe74) | 0 (Tyr336) | 62±3 | 1.32±0.07 | ||
| +9 (Tyr345) | 69±2 | 0.33±0.04 | |||
| 0 (Gln79) | −19 (Asp317) | <5 | n.d. | ||
| −8 (Leu328) | 53±1 | 4.16±0.13 | |||
| 0 (Tyr336) | 63±1 | 2.24±0.16 | |||
| +9 (Tyr345) | 77±2 | 0.47±0.03 | cGi-500 | ||
| +18 (Asn354) | 41±5 | 0.37±0.01 | |||
| +28 (Asp364) | 23±3 | 0.24±0.02 | |||
| −6 (Thr85) | −8 (Leu328) | 58±2 | 5.64±0.40 | cGi-6000 | |
| 0 (Tyr336) | 72±2 | 3.06±0.47 | cGi-3000 | ||
| +9 (Tyr345) | 74±3 | 0.75±0.21 | |||
| −17 (Glu96) | 0 (Tyr336) | 22±3 | 0.49±0.02 | ||
| +9 (Tyr345) | 25±3 | 0.13±0.01 | |||
| cNMP-BD A (Gln79–Asn228) | +5 (Phe74) | +5 (Leu233) | <5 | n.d. | |
| 0 (Gln79) | +5 (Leu233) | <5 | n.d. | ||
| +16 (Asn244) | <5 | n.d. | |||
| +28 (Asp256) | <5 | n.d. |