TABLE 2.
SNAP-25 alanine mutants
Residues determined to be important for Gβγ binding to SNAP-25 peptides were successively introduced into the native SNAP-25 sequence. Listed are the names given to each SNAP-25 mutant with the corresponding list of residues mutated to alanine. Residues in boldface are the new mutated residue added to the previously made mutant SNAP-25. Max is the maximum fluorescence enhancement (F1/F0) of the nonlinear regression for the each mutant, normalized to the maximum enhancement for wild-type SNAP-25. The N4A mutant contains only the four alanine mutations near the N terminus of the SNARE complex. Data are means (± S.E.).
| Mutant Name | Residues of SNAP-25 Mutated | Log EC50 | EC50 | Max |
|---|---|---|---|---|
| WT | N.A. | −6.45 (± 0.20) | 3.5 × 10−7, (2.2 × 10−7–5.6 × 10−7) | 100 (± 15) |
| 2A | R198A, K201A | −6.18 (± 0.13) | 6.6 × 10−7, (4.9 × 10−7–8.9 × 10−7) | 96 (± 10) |
| 3A | E62A, R198A, K201A | −5.93 (± 0.12) | 1.2 × 10−6, (8.9 × 10−7–1.5 × 10−6) | 122 (± 16) |
| 4A | E62A, D99A, R198A, K201A | −6.12 (± 0.13) | 7.6 × 10−7, (5.6 × 10−7–1.0 × 10−6) | 71 (± 8) |
| 5A | E62A, D99A, K102A, R198A, K201A | −6.08 (± 0.15) | 8.3 × 10−7, (5.9 × 10−7–1.7 × 10−6) | 70 (± 9) |
| 6A | E62A, D99A, K102A, R135A, R198A, K201A | −5.97 (± 0.08) | 1.1 × 10−6, (7.4 × 10−7–1.2 × 10−6) | 88 (± 8) |
| 7A | E62A, D99A, K102A, R135A, R136A, R198A, K201A | −6.12 (± 0.09) | 7.6 × 10−7, (6.2 × 10−7–8.9 × 10−7) | 33 (± 3) |
| 8A | E62A, D99A, K102A, R135A, R136A, R142A, R198A, K201A | −5.87 (± 0.12) | 1.3 × 10−6, (1.02 × 10−6–1.8 × 10−6) | 20 (± 3) |
| 9A | E62A, D99A, K102A, R135A, R136A, R142A, R161A, R198A, K201A | −6.36 (± 0.23) | 4.4 × 10−7, (2.6 × 10−7–7.4 × 10−7) | 25 (± 4) |
| N4A | R135A, R136A, R142A, R161A | −6.69 (± 0.14) | 2.0 × 10−7, (1.5 × 10−7–2.8 × 10−7) | 44 (± 3) |
N.A., not applicable.