Table 2.

Free energies of the Try-to-pTyr mutation in water and in IGF-1RK, determined by the TI method and the three-step transformation process presented in Figure S1.^a The energy is in the unit of kcal/mol.

	$\mathrm{\Delta }{\mathbf{\text{F}}}_{\mathbf{\text{TI}}}^{\mathbf{\text{CSC}}}$a				$\mathrm{\Delta }{\mathbf{\text{F}}}_{\mathbf{\text{TI}}}^{\mathbf{\text{GSC}}}$b
	Total c	Step 1	2	3	Totalc	Step 1	2	3
ΔF (water) d	−205.99 ± 0.16	15.41 ± 0.02	5.33 ± 0.12	−226.73 ± 0.11	−206.09 ± 0.16	17.92 ± 0.02	11.46 ± 0.11	−235.47 ± 0.11
ΔF (IGF-1RK) e	−212.24 ± 0.27	14.77 ± 0.03	3.82 ± 0.19	−230.83 ± 0.18	−212.87 ± 0.29	21.25 ± 0.04	13.01 ± 0.17	−247.13 ± 0.23
ΔΔF (Stability) f	−6.25				−6.78

^aIn the CSC protocol, Step 1 removes the charge of the solute, Step 2 transforms the vdW parameters of the solute to those of the transformed solute with CSC and Step 3 introduces the charge of the transformed solute, respectively (Figure S1a).

^bIn the GSC protocol, Step 1 removes the charge and the vdW interactions of the solute with the introduction of the solute GSC potential, Step 2 transforms the solute GSC potential to the transformed solute’s GSC potential and Step 3 introduces the charge and the vdW interactions of the transformed solute with the removal of the GSC potential, respectively (Figure S1b).

^c“Total” refers the total FE change of the entire transformation.

^dFree energies of the mutation in water. Each step was achieved with 19 λ simulations.

^eFree energies of the mutation in the protein IGF-1RK. Each step was achieved with 11 λ simulations.

^fThe relative free energy between protein and water, i.e., ∆∆F = ∆F (IGF-1RK) −∆F (water), in each step as well as the entire transformation.