Table 1. Comparison of biochemical parameters, velocities, rate, and equilibrium constants measured in experiments with human cytoskeletal γ-actin and variant p.E334Q.
| Parameter | γ-actin | p.E334Q | 1:1 mixture |
|---|---|---|---|
| IC50 (nM) (DNase inhibition) | 22.7 ± 0.7 | 25.7 ± 0.6 (*1.1-fold ↑) |
– |
| TM (°C) (Mg2+-ATP-G-actin) | 56.77 ± 0.24 | 56.19 ± 0.30 (ns) |
– |
| TM (°C) (Mg2+-ADP-F-actin) | 65.00 ± 0.76 | 64.83 ± 0.25 (ns) |
– |
| k-T (s–1) (Mg2+-ε-ATP) | 0.0473 ± 0.0013 | 0.0972 ± 0.0019 (**** 2.1-fold ↑) |
– |
| k-D (s–1) (Mg2+-ε-ADP) | 0.2155 ± 0.0017 | 0.3199 ± 0.0027 (**** 1.5-fold ↑) |
– |
| F-actin ATPase (s–1) | 0.00089 ± 0.00005 | 0.00088 ± 0.00004 (ns) |
– |
| kobs (s–1) bulk polymerization rate (Δpyr-fluorescence) | 0.0140 ± 0.0004 | 0.0129 ± 0.0008 (***1.1-fold ↓) |
0.0134 ± 0.0006 (*1.04-fold ↓) |
| kobs (s–1) + 10 nM Arp2/3 bulk polymerization rate (Δpyr-fluorescence) | 0.6441 ± 0.0163 | 0.5458 ± 0.02256 (**1.2-fold ↓) |
0.6162 ± 0.0776 (ns) |
| robs (nm s–1) rate of filament elongation (TIRF-M) | 12.6 ± 0.3 | 11.1 ± 0.3 (**** 1.1-fold ↓) |
– |
|
kobs (s–1) rate of filament disassembly (Δpyr-fluorescence) |
0.0022 ± 0.0002 | 0.0029 ± 0.0003 (**** 1.3-fold ↑) |
0.0023 ± 0.0002 (ns) |
| kc (nM–1 s–1) + cofilin rate of filament disassembly (Δpyr-fluorescence) | 3.42 × 10–4 ± 0.22 × 10–4 | 0.81 × 10–4 ± 0.08 × 10–4
(**** 4.2-fold ↓) |
1.54 × 10–4 ± 0.11 × 10–4 (*** 2.2-fold ↓) |
| v (nm s–1) sliding velocity on NM2A-HMM | 195.3 ± 5.0 | 39.1 ± 3.2 (**** 5.0-fold ↓) |
131.2 ± 10.0 (*** 1.5-fold ↓) |
| v (nm s–1) sliding velocity on Myo5A-HMM at optimal myosin density | 315.1 ± 28.9 | 190.4 ± 46.9 (**** 1.7-fold ↓) |
– |
| Kapp (µM) (NM2A-HMM) | 3.20 ± 0.74 | 2.89 ± 0.49 (ns) |
– |
| kcat (s–1) (NM2A-HMM) | 0.097 ± 0.002 | 0.076 ± 0.005 ( *1.3-fold ↓) |
– |
| k-A (s–1) (NM2A-2R) | 8.0 × 10–5 ± 3.3 × 10–6 | 4.5 × 10–4 ± 6.7×10–6 (**** 5.6-fold ↑) |
2.3 × 10–4 ± 1.3 × 10–5(***2.9-fold ↑) |
| k-A (s–1) (NM2C-2R) | 3.1 × 10–4 ± 5.0 × 10–6 | 1.7 × 10–3 ± 5.8 × 10–5 (***5.6-fold ↑) |
6.7 × 10–4 ± 1.5×10–5 (***2.2-fold ↑) |
| KA (nM) (NM2A-2R) | 1.7 ± 0.3 | 16.9 ± 0.3 (**9.9-fold ↑) |
– |
| KDA (nM) (NM2A-2R) | 122.6 ± 11.4 | 353.0 ± 20.6 (**2.9-fold ↑) |
– |
| Coupling (KDA/KA) (NM2A-2R) |
72.1 | 20.9 (**** 3.5-fold ↓) |
– |
| 1/K1 (µM) (NM2A-2R) | 812.1 ± 82.7 | 863.8 ± 91.6 (ns) |
– |
| k+2 (s–1) (NM2A-2R) | 461.7 ± 18.6 | 435.6 ± 16.5 (ns) |
– |
| K1k+2 (µM–1 s–1) (NM2A-2R) | 0.276 ± 0.002 | 0.272 ± 0.005 (ns) |
– |
Bold entries indicate the observed difference between wt and mutant values. ns = p> 0.05, * p≤ 0.05 , ** p≤ 0.01, *** p ≤ 0.001, **** p≤ 0.0001.