Table 3. Predicted molecular weights and rotational correlation times for Venus, Venus-LC3B, and Venus-LC3B mutants based on the FPFA diffusion measurements in HEK 293 cell extracts under basal conditions, and after incubation with 100 μM chloroquine (CQ) for 2 h.
| Construct | Monomer MW (kDa) | Basal predicted MW (kDa)* | CQ predicted MW (kDa)* | Basal percent species (%) | Basal rotation correlation time (ns) | CQ rotation correlation time (ns) |
|---|---|---|---|---|---|---|
| Venus | 27 | N/A | N/A | N/A | 12.3 ± 0.2 (4) | N/A |
| Venus-LC3B | 45 | 500 ± 100 (10) | 480 ± 90 (6) | N/A | 24.9 ± 0.6 (10) | 25.0 ± 0.3 (6) |
|
Fast Venus-LC3B (F52A/L53A) |
45 | 300 ± 100 (7) | 190 ± 70 (3) | 70 ± 4 (4) | 25.6 ± 0.3 (7) | 25.5 ± 0.1 (4) |
|
Slow Venus-LC3B (F52A/L53A) |
45 | 5 × 104 ± 5 × 104 (7) | N/A | 30 ± 4 (4) | N/A | N/A |
| Venus-LC3B (R70A) | 45 | 190 ± 50 (7) | 260 ± 30 (3) | N/A | 24.3 ± 0.6 (7) | 23.9 ± 0.1 (3) |
| Venus-LC3B (G120A) | 45 | 600 ± 90 (7) | 330 ± 40 (7) | N/A | 24.8 ± 0.3 (7) | 25.3 ± 0.5 (7) |
*Calculated assuming diffusing species has a spherical shape; mean ± 95% CI (N, # extracts); CQ, chloroquine.