TABLE 1.
Sedimentation velocity analysis of NEMO ZF interaction with mono-Ub or various forms or di-Ub (linear and Lys-48- and Lys-63-linked)
| Protein | Sedimentation coefficient | f/f0 | Molecular mass | Complex stoichiometry ZF-Ubi |
|---|---|---|---|---|
| S* | kDa | |||
| F-ZF (150 μm) | 1.0 ± 0.1 | 1.2 | 4.7 ± 1 | |
| Mono-Ub (100 μm) | 1.3 ± 0.1 | 1.06 | 8.4 ± 0.4 | |
| Lys-48-di-Ub (100 μm) | 1.8 ± 0.1 | 1.07 | 15.4 ± 2 | |
| Lys-63-di-Ub (100 μm) | 1.9 ± 0.1 | 1.06 | 15.0 ± 2 | |
| Met-1-di-Ub (100 μm)a | 1.9 ± 0.1 | 1.12 | 16.1 ± 1 | |
| F-ZF + mono-Ub (300:100 μm)b,c | 1.9 ± 0.1 (1.05) | 1.5 | 20.8 ± 2.3 | 1:2 |
| F-ZF + Lys-48-di-Ub (150:100 μm)b | 2.3 ± 0.1 (1.0) | 1.22 | 20.3 ± 2.2 | 1:1 |
| F-ZF + Lys-63-di-Ub (150:100 μm)b | 2.3 ± 0.1 (0.95) | 1.16 | 19.0 ± 2.3 | 1:1 |
| F-ZF + Met-1-di-Ub (150:100 μm)b | 2.2 ± 0.1 (1.01) | 1.26 | 20.8 ± 2.0 | 1:1 |
a A heavier species with a sedimentation coefficient of 4.4 ± 0.5 S and a frictional coefficient (f/f0) of 1.12 was observed when Met-1-diUb was analyzed alone. This species of 59 ± 8 kDa was compatible with a trimer of Met-1-diUb.
b The sedimentation coefficients measured for the free state of fluorescein N-terminal-labeled NEMO are indicated in parentheses.
c A minor complex of ZF·monoUb with a stoichiometry of 2:4 (44.3 kDa) was also detected under these experimental conditions.