Table 3. Measures of Fluorescence Lifetime of P450:eGFP and FRET Evaluation of Associations between P450:eGFP and Soluble Enzymes:mRFP1.
| Expressed Proteins |
Fluorescence Lifetime (ns) |
FRET Efficiency |
|||||
|---|---|---|---|---|---|---|---|
| Donor | Acceptor | n | Untagged Proteins | Average | sd | % | sd |
| CYP73A5:eGFP |
/ | / | 121 | 2.05 | 0.09 | – | – |
| mRFP1 | / | 30 | 1.97 | 0.08 | 3.6 | 0.2 | |
| mRFP1 | CYP98A3/HCT /4-CL1 | 10 | 2.20 | 0.08 | 0.0 | 0.4 | |
| mRFP1:4-CL1 | / | 17 | 2.10 | 0.05 | 0.0 | 0.1 | |
| CYP98A3/HCT | 20 | 1.93 | 0.07 | 5.7 | 0.3 | ||
| 4-CL1:mRFP1 | / | 41 | 1.94 | 0.07 | 5.3 | 0.3 | |
| CYP98A3/HCT | 16 | 2.02 | 0.05 | 1.4 | 0.1 | ||
| HCT:mRFP1 | / | 17 | 1.99 | 0.03 | 2.9 | 0.1 | |
| CYP98A3/4-CL1 | 17 | 1.88 | 0.06 | 8.6 | 0.5 | ||
| mRFP1:HCT | / | 17 | 2.06 | 0.07 | 0.0 | 0.0 | |
| CYP98A3/4-CL1 | 17 | 1.84 | 0.07 | 10.3 | 0.6 | ||
| CYP98A3:eGFP | / | / | 112 | 2.12 | 0.15 | – | – |
| mRFP1 | / | 17 | 2.00 | 0.14 | 5.5 | 0.5 | |
| mRFP1 | CYP73A5/HCT/4-CL1 | 11 | 2.33 | 0.08 | 0.0 | 0.8 | |
| HCT:mRFP1 | / | 25 | 2.18 | 0.12 | 0.0 | 0.3 | |
| CYP73A5/4-CL1 | 15 | 1.73 | 0.09 | 18.2 | 1.6 | ||
| mRFP1:HCT | / | 26 | 1.90 | 0.12 | 10.3 | 1.0 | |
| CYP73A5/4-CL1 | 22 | 1.89 | 0.12 | 10.5 | 1.0 | ||
| 4-CL1:mRFP1 | / | 37 | 2.06 | 0.14 | 2.7 | 0.3 | |
| CYP73A5/HCT | 26 | 1.92 | 0.16 | 9.4 | 1.0 | ||
| mRFP1:4-CL1 | / | 27 | 2.06 | 0.19 | 2.4 | 0.3 | |
| CYP73A5/HCT | 22 | 1.86 | 0.21 | 12.2 | 1.6 | ||
Values of P450:eGFP fluorescence lifetimes were obtained by FLIM. FRET efficiency is calculated by comparison with eGFP fluorescence lifetime measured in a control experiment in absence of the mRFP1 acceptor protein. To determine the contribution of the different partners to the interaction between each P450 and soluble protein, the other proteins were expressed without fluorescent tags. n, number of independent cells analyzed. Slash indicates no acceptor; dash indicates no value; bold value indicates value higher than controls. See Supplemental Table 2 online for FLIM measured between soluble proteins and Supplemental Figure 4 online for dot blot confirmation of protein expression.