Table 2.
In vitro measurement of dissociation constants of Mex67 and Rad23-derived GST and His6 fusion proteins of the respective UBA domains for mono- and Lys48-linked tetraubiquitin (K48-Ub4)
| Recombinant protein | KD for mono-Ub (μM) | KD for K48-Ub4 (μM) |
|---|---|---|
| GST-UBA2-Rad23 | 19 ± 2 | 7.8 ± 1.2 |
| His-UBA2-Rad23 | 12.9 ± 3.2 | 6.1 ± 0.2 |
| GST-UBA-Mex67 | 400 | >300 |
| His-UBA-Mex67 | 400 | >400 |
| UBA-Mex67 | Not determined | >300 |
| GST-UBAΔH4-Mex67 | 63 ± 4 | 18 ± 2.4 |
| UBA-Mex67(V597A) | Not determined | >300 |
| UBA-Mex67(F596A) | Not determined | 68 ± 5 |
| His-UBA-Mex67:GST-Hpr1 | Not detectable | 7.5 ± 3 |
| GST-Hpr1 | No binding | No binding |
| GST-Mex67 | 12 ± 3 | 6.2 ± 0.6 |
| GST-Mex67ΔUBA | 300–400 | 300 |
| GST-Mex67ΔH4 | 13 ± 2.1 | 3.7 ± 1.2 |
The association with ubiquitin was measured in vitro by monitoring the fluorescence enhancement of fluorescently labeled ubiquitin.