PROLINE CIS-TRANS ISOMERIZATION IN STAPHYLOCOCCAL NUCLEASE - MULTI-SUBSTATE FREE-ENERGY PERTURBATION CALCULATIONS

Staphylococcal nuclease A exists in two folded forms that differ in th e isomerization state of the Lys 116-Pro 117 peptide bond. The dominan t form (90% occupancy) adopts a cis peptide bond, which is observed in the crystal structure. NMR studies show that the relatively small dif ference in free energy between the cis and trans forms (Delta G(cis--> trans) approximate to 1.2 kcal/mol) results from large and nearly comp ensating differences in enthalpy and entropy Delta H-cis-->trans appro ximate to Delta TScis-->trans approximate to 10 kcal/mol). There is ev idence from X-ray crystal structures' that the structural differences between the cia and the trans forms of nuclease are confined to the co nformation of residues 112-117, a solvated protein loop. Here, we obta in a thermodynamic and structural description of the conformational eq uilibrium of this protein loop through an exhaustive conformational se arch that identified several substates followed by free energy simulat ions between the substates. By partitioning the search space into conf ormational substates, we overcame the multiple minima problem in this particular case and obtained precise and reproducible free energy valu es. The protein and water environment was implicitly modeled by approp riately chosen nonbonded terms between the explicitly treated loop and the rest of the protein. These simulations correctly predicted a smal l free energy difference between the cia and trans forms composed of l arger, compensating differences in enthalpy and entropy. The structura l predictions of these simulations were qualitatively consistent with known X-ray structures of nuclease variants and yield a model of the u nknown minor trans conformation.