Table 1.
ZASP and Subdomains: Some Biophysical Parameters
| Mw (kDa)c |
|||||||
|---|---|---|---|---|---|---|---|
| proteina | His-tagb | sequence | AUC | speedd (rpm, × 103) | ν-bare (g/cm3) | A280f | affinityg Kd (M) |
| ZASP-LΔex10 | N24 | 69.37 | 60.0 (±1.3) | 14.5 | 0.724 | 0.95 | 3.9 × 10−8 |
| ZASP-S WT | N21 | 16.5 | 2.0 × 10−9 | ||||
| ZASP-S WT | none | 31.21 | 29.5 (±0.2) | 18.5 | 0.730 | 0.77 | 6.2 × 10−8 2.5 × 10−8h |
| ZASP-S 147 | none | 31.5 (±0.8) | 18.5 | 7.6 × 10−8 | |||
| ZASP-S 165 | none | 27.3 (±0.3) | 18.5 | 4.4 × 10−8 | |||
| ZASP-S 268 | none | 29.18 | 2.4 × 10−8 | ||||
| ZASP-S 1−251 | none | 27.00 | 0.68 | 2.5 × 10−8 | |||
| ABRΔ10 WT | N34 | 24.17 | 26.0 (±1.3) | 18.5 | 0.718 | 0.58 | 4.0 × 10−7 |
| ABRΔ10 147 | N34 | 33.6 (±0.8) | 18.5 | 2.6 × 10−7 | |||
| ABRΔ10 165 | N34 | 25.0 (±2.0) | 18.5 | 1.0 × 10−6 | |||
| ABRΔ10 WT | N21 | 22.80 | 24.4 (±1.0) | 20.0 | 0.718 | 0.58 | 4.6 × 10−7 3.6 × 10−8i |
| ABR+10 WT | N21 | 28.62 | 28.9 (±0.2) | 18.5 | 0.720 | 0.50 | 1.0 × 10−5 |
The proteins indicated are either wild-type sequence (WT) or the mutants A147T, A165V and R268C.
N-terminal His-tags: N21 = MGSSHHHHHHSSGLVPRGSHM, N24 = HHHHHHDYDIPTTENLYFQGAMGS, N34 = MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRIP.
The molecular weights of the constructs as derived from the gene sequence and determined (with error indicated) under native conditions by sedimentation equilibrium (AUC).
The speeds used for AUC analysis.
The partial specific volumes (ν-bar) were calculated from the amino acid compositions.
Absorbance was calculated from amino acid composition: values indicated are A280 nm of 1 mg/mL protein using a 1 cm path-length cell.
The binding of the proteins to immobilized G-actin was measured using SPR (Biacore). The binding affinities are given as dissociation constants (Kd) and are in molar (M) units.
Affinity determined with ZASP-S immobilized and G-actin as the analyte.
Affinity determined by titration calorimetry.