Table 1.
γ-corrected smFRET efficiencies between fluorescent dye labels attached to complexin-1 (Cpx) mutants (WT, wildtype; SC, superclamp; NC, no-clamp) and ternary SNARE complex obtained from smFRET measurements. The γ-factors for the Cpx mutants (Cpx WT: 1.66, Cpx SC: 1.62, Cpx NC: 1.42) were empirically estimated, and the corresponding mean values were used to correct the corresponding smFRET efficiency histogram shown in Figure 3A,B using Equation (4) as described in the Materials and methods. The two FRET efficiencies shown in the table are the peak positions of two Gaussian functions that were fit to the γ-corrected smFRET efficiency histograms, and the error bounds are standard deviations of the peak positions. For comparison, FRET efficiencies were calculated from the specified crystal structures (PDB IDs: 3RK3, 3RL0, and 1KIL, see Materials and methods for the calculation of FRET efficiencies from crystal structures). Cpx was labeled with FRET donor dye (Alexa 555) molecule at residue position 24 and syntaxin-1A was labeled with FRET acceptor dye (Alexa 647) molecule at residue position 249.
| γ-corrected smFRET efficiency |
FRET efficiency calculated from the specified crystal structures |
|
|---|---|---|
| Cpx WT | 0.19 ± 0.12 (trans) 0.83 ± 0.15 (cis) |
0.13 (PDB ID: 3RK3, 3RL0) 0.49 (PDB ID: 1KIL) |
| Cpx SC | 0.28 ± 0.18 (trans) 0.83 ± 0.14 (cis) |
|
| Cpx NC | 0.24 ± 0.13 (trans) 0.81 ± 0.12 (cis) |