Table 1. FRET constructs derived from the GAF domains of PDE5.
Shown are the relative changes in CFP/YFP emission ratio of the constructs derived from the PDE5 GAF domains determined in vitro as described in the Materials and methods section. The borders of the constructs containing either GAF A+B or GAF A only (see Figure 1) are stated relative to the border of the GAF domains (left column) of human PDE5a (GenBank® accession number NM_001083.2). The first and the last amino acid (aa) relative to the PDE5 holoenzyme are given in parentheses. n.d., not determined because the constructs displayed very low expression. The numbers in bold type highlight the constructs displaying FRET changes.
| Indicator construct | N-terminal elongation (first aa) | C-terminal elongation (last aa) | Change in CFP/YFP emission ratio (%) |
|---|---|---|---|
| GAF A+B (Asp144–Ala517) | +60 (Gln84) | +10 (Ser527) | 50 |
| +51 (Val93) | −23 (Ala494) | <5 | |
| −10 (Gln507) | <5 | ||
| −2 (Ala515) | 80 | ||
| +10 (Ser527) | 40 | ||
| +27 (Ala544) | 30 | ||
| +44 (Lys100) | −23 (Ala494) | <5 | |
| −16 (Leu501) | <5 | ||
| −10 (Gln507) | <5 | ||
| −2 (Ala515) | <5 | ||
| +10 (Ser527) | <5 | ||
| +12 (His529) | <5 | ||
| +30 (Val114) | −2 (Ala515) | <5 | |
| +10 (Ser527) | <5 | ||
| +21 (Ser123) | −2 (Ala515) | <5 | |
| +10 (Ser527) | <5 | ||
| +10 (Met134) | −31 (Arg486) | <5 | |
| −2 (Ala515) | <5 | ||
| +10 (Ser527) | <5 | ||
| 0 (Asp144) | −16 (Leu501) | 7 | |
| +12 (His529) | <5 | ||
| −10 (Glu154) | −31 (Arg486) | n.d. | |
| GAF A (Asp144–Ile312) | +60 (Gln84) | +14 (Glu326) | <5 |
| +51 (Val93) | −19 (Gly293) | <5 | |
| +2 (Leu314) | <5 | ||
| +14 (Glu326) | <5 | ||
| +35 (Leu347) | <5 | ||
| +21 (Ser123) | +2 (Leu314) | <5 | |
| +14 (Glu326) | <5 | ||
| 0 (Asp144) | +2 (Leu314) | <5 | |
| +14 (Glu326) | <5 | ||
| +26 (Ser338) | <5 | ||
| +35 (Leu347) | <5 | ||
| −21 (Val165) | +2 (Leu314) | n.d. | |
| +14 (Glu326) | <5 |