Proline cis‐trans isomerization in staphylococcal nuclease: Multi‐substate free energy perturbation calculations

Staphylococcal nuclease A exists in two folded forms that differ in the isomerization state of the Lys 116‐Pro 117 peptide bond. The dominant form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small difference in free energy between the cis and trans forms (δDLGcis‐trans ≈ 1.2 kcal/mol) results from large and nearly compensating differences in enthalpy and entropy (ΔGcis‐trans (ΔTScis‐trans ≈ 10 kcal/mol). There is evidence from X‐ray crystal structures that the structural differences between the cis and the trans forms of nuclease are confined to the conformation of residues 112–117, a solvated protein loop. Here, we obtain a thermodynamic and structural description of the conformational equilibrium of this protein loop through an exhaustive conformational search that identified several substates followed by free energy simulations between the substates. By partitioning the search space into conformational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy values. The protein and water environment was implicitly modeled by appropriately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a small free energy difference between the cis and trans forms composed of larger, compensating differences in enthalpy and entropy. The structural predictions of these simulations were qualitatively consistent with known X‐ray structures of nuclease variants and yield a model of the unknown minor trans conformation.

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