Table 1.
Overview of AMPfret constructs tested for AMP responses
| AMPfret construct | Organization | YFP peaka | FRET ratiob | Distances (Å)c | FRET changed |
|---|---|---|---|---|---|
| AMPK 221 | α2 _ β2 _ γ1 | / | / | / | / |
| AMPfret A (AMPfret pre-2.0) | α2-CFP _ β2-YFP _ γ1 | ++ | 1.444 | 13.8 | + |
| AMPfret B | CFP-α2 _ β2-YFP _ γ1 | ± | 0.492 | 78.9 | − |
| AMPfret C (AMPfret 1.0) | α2-CFP _ β2 _ γ1-YFP | + | 0.639 | 59.8 | + |
| AMPfret D | α2-CFP _ β2 _ YFP-γ1 | ± | 0.513 | 62.7 | − |
| AMPfret E | CFP-α2 _ β2 _ γ1-YFP | − | / | 78.5 | − |
| AMPfret F (AMPfret CTL) | CFP-α2 _ β2 _ YFP-γ1 | − | / | 72.7 | − |
| AMPfret G | α2 _ β2-YFP _ γ1-CFP | ± | 0.524 | 50.0 | − |
| AMPfret H | α2 _ YFP-β2 _ γ1-CFP | ± | 0.464 | (63.3) | − |
| AMPfret I | α2 _ β2-YFP _ CFP-γ1 | + | 0.637 | 53.1 | − |
| AMPfret J | α2 _ YFP-β2 _ CFP-γ1 | ± | 0.559 | (58.2) | − |
| AMPfret K | α2-CFP _ YFP-β2 _ γ1 | + | 0.648 | (67.8) | ± |
| AMPfret L | CFP-α2 _ YFP-β2 _ γ1 | − | / | (17.7) | − |
| AMPfret 2.0 | α2-helix-CFP _ β2-YFP _ γ1 | ++ | 2.032 | 19.0 ± 11.6 | ++ |
| AMPfret 1.1 | α2-mseCFPΔ11 _ β2 _ γ1-cpVenus | + | 0.727 | 55.0 | + |
| AMPfret 2.1 | α2-helix-mseCFPΔ11 _ β2-cpVenus _ γ1 | ++ | 2.026 | 19.0 ± 11.6 | ++ |
aYFP peak visibility annotated as: ++ strong, + detectable, ± shoulder, – absent
bFRET ratio (YFP/CFP) at baseline
cDistances between tagged termini determined from PDB IDs: 5ISO and 4CFF26. In brackets: distances involving the β N-ter lacking in these structures. For AMPfret 2.0/2.1: a range is given, since orientation of the added α-helix linker could not be predicted
dChange of FRET ratio upon AMP addition annotated as: ++ strong, + average, ± detectable, – non-detectable