Table 3.
Fluorescence anisotropy measurements of Alexa 555 or Alexa 647 dyes linked to the specified residues in the cytoplasmic domains of syntaxin-1 (SX) or synaptobrevin-2 (SB), either in isolation or as part of the ternary SNARE complex, in the presence and absence of 1 μM full-length wildtype complexin-1 (Cpx [1-134]). The numbers after 'SX' and 'SB' are the residue positions of the corresponding labeling sites. The formation of ternary SNARE complex and the presence of complexin-1 did not substantially affect the fluorescence anisotropy. Shown are means ± SD for n = 3 replicates.
| Constructs | Steady state anisotropy (r) |
|---|---|
| Alexa 555 | 0.20 ± 0.06 |
| Alexa 555 (linked to SB 91) | 0.26 ± 0.01 |
| Alexa 555 (linked to SB 91, in ternary complex) | 0.31 ± 0.01 |
| Alexa 555 (linked to SB 91, in ternary complex) + 1 μM Cpx | 0.32 ± 0.03 |
| Alexa 555 (linked to SB 82) | 0.27 ± 0.01 |
| Alexa 555 (linked to SB 82, in ternary complex) | 0.27 ± 0.02 |
| Alexa 555 (linked to SB 82, in ternary complex) + 1 μM Cpx | 0.28 ± 0.06 |
| Alexa 555 (linked to SB 82, in ternary complex) + 10 μM Cpx | 0.28 ± 0.1 |
| Alexa 555 (linked to SB 28) | 0.24 ± 0.03 |
| Alexa 555 (linked to SB 28, in ternary complex) | 0.32 ± 0.01 |
| Alexa 555 (linked to SB 28, in ternary complex) + 1 μM Cpx | 0.32 ± 0.004 |
| Alexa 647 | 0.15 ± 0.08 |
| Alexa 647 (linked to SX 259) | 0.24 ± 0.09 |
| Alexa 647 (linked to SX 259, in ternary complex) | 0.27 ± 0.1 |
| Alexa 647 (linked to SX 259, in ternary complex) + 1 μM Cpx | 0.27 ± 0.1 |
| Alexa 647 (linked to SX 249) | 0.24 ± 0.02 |
| Alexa 647 (linked to SX 249, in ternary complex) | 0.26 ± 0.1 |
| Alexa 647 (linked to SX 249, in ternary complex) + 1 μM Cpx | 0.27 ± 0.08 |
| Alexa 647 (linked to SX 249, in ternary complex) + 10 μM Cpx | 0.28 ± 0.1 |
| Alexa 647 (linked to SX 193) | 0.27 ± 0.1 |
| Alexa 647 (linked to SX 193, in ternary complex) | 0.26 ± 0.08 |
| Alexa 647 (linked to SX 193, in ternary complex) + 1 μM Cpx | 0.27 ± 0.08 |