DETERMINATION OF THE DIFFERENTIAL-EFFECTS OF HYDROGEN-BONDING AND WATER RELEASE ON THE BINDING OF FK506 TO NATIVE AND TYR82-]PHE82 FKBP-12 PROTEINS USING FREE-ENERGY SIMULATIONS

We use the thermodynamic integration technique to calculate the free e nergy associated with the Tyr82 --> Phe82 mutation (Y82F) in the prote in FKBP-12, both free and bound to known inhibitor FK506 (tacrolimis). We find that the net difference in free energy for the two changes is 0.85 kcal/mol, with the binding of FK506 relatively more favorable fo r the native protein than the mutant. This net energy compares very fa vorably with the experimentally measured value of 0.60 kcal/mol. The r esults indicate that the relatively better binding of FK506 to the nat ive protein is driven by the favorable entropy associated with the rel ease of water molecules from the protein when the ligand binds. For a variety of reasons, modest size of the system, smallness of the change being examined, rapid convergence of the ensemble that needs to be de termined and use of statistical estimates to control sampling, we have been able to carry out atypically reliable and reproducible free ener gy calculations for this protein system. Free energy changes for the t wo simulations (Y82F FKBP-12/FK506 and Y82F FKBP-12) have been calcula ted a total of eight times each, to compare a variety of different met hodological choices and to ensure that the results are statistically s ignificant. Detailed analysis of the free energy results has been carr ied out, and indicates that even when applicable, deconvolution of the total free energy into components can be very difficult, that the sta tistical error estimates can give a reasonable bound on the error in a simulation, and that one must be careful to use the same simulation p rotocol in all simulations being compared.