Binding parameters on microtubules of full-length, truncated, and chimeric versions of VASH1 and VASH2 enzymes in complex with SVBP
| Experiment (medium) . | Ionic strength (mM) . | Microtubule . | Recombinant protein (concentration) . | Residence time τ (s) . | Binding frequency (min-1.nM−1.µm−1) . |
|---|---|---|---|---|---|
| A (BRB40, 50 mM KCl) | 140 | Tyr-MTs | deadV1_FL (50 pM) | 1.2 | 80.4 |
| deadV1_Nt + CD (50 pM) | - | No binding | |||
| deadV1_CD (50 pM) | 1.4 | 70.7 | |||
| deadV1_CD + Ct (50 pM) | >7.7 | 21.1 | |||
| deadV2_FL (50 pM) | >17.1 | 16.2 | |||
| deadV2_Nt + CD (50 pM) | >4.0 | 35.9 | |||
| deadV2_CD (50 pM) | 1.2 | 85.1 | |||
| deadV2_CD + Ct (50 pM) | >10.4 | 10.4 | |||
| B (BRB40, 50 mM KCl) | 140 | Tyr-MTs | deadV1_FL (50 pM) | 1.2 | 54.1 |
| deadV1_FL(NtV2) (50 pM) | >9.9 | 31.0 | |||
| deadV2_FL (50 pM) | >13.4 | 19.1 | |||
| deadV2_FL(NtV1) (50 pM) | >6.5 | 18.1 | |||
| C (BRB40, 50 mM KCl) | 140 | Tyr-MTs | V1_FL (25 pM) | 1.2 | 96.7 |
| V1_FL(NtV2) (25 pM) | >10.7 | 20.8 | |||
| V2_FL (25 pM) | >12.9 | 22.9 | |||
| V2_FL(NtV1) (25 pM) | >3.9 | 42.4 |
| Experiment (medium) . | Ionic strength (mM) . | Microtubule . | Recombinant protein (concentration) . | Residence time τ (s) . | Binding frequency (min-1.nM−1.µm−1) . |
|---|---|---|---|---|---|
| A (BRB40, 50 mM KCl) | 140 | Tyr-MTs | deadV1_FL (50 pM) | 1.2 | 80.4 |
| deadV1_Nt + CD (50 pM) | - | No binding | |||
| deadV1_CD (50 pM) | 1.4 | 70.7 | |||
| deadV1_CD + Ct (50 pM) | >7.7 | 21.1 | |||
| deadV2_FL (50 pM) | >17.1 | 16.2 | |||
| deadV2_Nt + CD (50 pM) | >4.0 | 35.9 | |||
| deadV2_CD (50 pM) | 1.2 | 85.1 | |||
| deadV2_CD + Ct (50 pM) | >10.4 | 10.4 | |||
| B (BRB40, 50 mM KCl) | 140 | Tyr-MTs | deadV1_FL (50 pM) | 1.2 | 54.1 |
| deadV1_FL(NtV2) (50 pM) | >9.9 | 31.0 | |||
| deadV2_FL (50 pM) | >13.4 | 19.1 | |||
| deadV2_FL(NtV1) (50 pM) | >6.5 | 18.1 | |||
| C (BRB40, 50 mM KCl) | 140 | Tyr-MTs | V1_FL (25 pM) | 1.2 | 96.7 |
| V1_FL(NtV2) (25 pM) | >10.7 | 20.8 | |||
| V2_FL (25 pM) | >12.9 | 22.9 | |||
| V2_FL(NtV1) (25 pM) | >3.9 | 42.4 |
(A) Related to Fig. 5, D and E. (B and C) Related to Fig. 6, A and B. A, B, and C represent experiments performed on different days. At least 10 microtubules were analyzed. See Materials and methods for detailed experimental conditions.