in silico
Phase 1 : In Silico Validation
- Objectif
- To computationally test whether the proposed electrostatic repulsion mechanism can produce a 4- to 8-fold increase in Kd, and to identify the minimal charge change required, before committing to expensive protein purification and single-molecule experiments.
- Coût estimé
- €500-2000
- Durée estimée
- 4-6 weeks
- Critères de réussite
- Computed Kd ratio (4E/WT) at 20% DOPS, 150 mM NaCl · ≥ 3.5 (to allow for simulation noise, but close to the predicted 4-8 fold) · (From BD simulation: Kd = k_off/k_on, averaged over 1000 trajectories per condition)
- Electrostatic binding free energy difference (ΔΔG) between WT and 4E · ΔΔG ≥ 1.5 kcal/mol (corresponding to ~4-fold Kd change at 25°C) · (From APBS: ΔΔG = ΔG(4E) - ΔG(WT) using the linearized Poisson-Boltzmann equation)
- Sensitivity: Kd ratio at 150 mM NaCl vs. 200 mM NaCl · Ratio changes by < 2-fold (i.e., the effect is not solely due to ionic strength artifacts) · (Repeat BD simulation at 200 mM NaCl and compare Kd ratio)
- On continue si
- Both (a) computed Kd ratio ≥ 3.5 and (b) ΔΔG ≥ 1.5 kcal/mol are satisfied.
- On arrête si
- Kd ratio < 2.0 OR ΔΔG < 0.5 kcal/mol, indicating the electrostatic mechanism is insufficient to explain the predicted effect.
- On réoriente si
- Kd ratio is 2.0-3.4 but ΔΔG ≥ 1.5 kcal/mol: consider that the 4E mutant may not fully mimic phosphorylation (e.g., charge distribution vs. phosphate geometry). Proceed to Phase 2 with a note to test both 4E and in vitro phosphorylated LIC1.
- Risques
- Coarse-grained BD simulation may not capture the flexibility of the intrinsically disordered region accurately.Probabilité : mediumParade : Use a more detailed all-atom MD simulation (e.g., GROMACS, 100 ns) for a subset of conditions to validate BD results. If resources limited, use a published force field for disordered proteins (e.g., ff14IDPSFF).
- The predicted Kd ratio may be highly sensitive to the dielectric constant at the membrane interface.Probabilité : mediumParade : Perform a sensitivity analysis with ε=4 (membrane interior) to ε=80 (bulk water) using a two-dielectric model in APBS. Report the range of ΔΔG values.
- No experimental structure of LIC1 C-terminus exists; AlphaFold2 may predict a structure that is not representative of the disordered state.Probabilité : highParade : Use an ensemble of random coil conformations generated by UCSF ChimeraX or PEP-FOLD3 and run BD simulations for each ensemble member. Average results over the ensemble.