Computational methodology for estimating changes in free energies of biomolecular association upon mutation. The importance of bound water in dimer-tetramer assembly for beta 37 mutant hemoglobins

The computational modeling program HINT (Hydropathic INTeractions), an empi rical hydropathic force field that includes hydrogen bonding, Coulombic, an d hydrophobic terms, was used to model the free energy of dimer-tetramer as sociation in a series of deoxy hemoglobin beta 37 double mutants. Five of t he analyzed mutants (beta 37W -> Y, beta 37W - A, beta 37W - G, beta 37W - E, and beta 37W - R) have been solved crystallographically and characterize d thermodynamically and subsequently made a good test set for the calibrati on of our method as a tool for free energy prediction. Initial free energy estimates for these mutants were conducted without the inclusion of crystal lographically conserved water molecules and systematically underestimated t he experimentally calculated loss in free energy observed for each mutant d imer-tetramer association. However, the inclusion of crystallographic water s, interacting at the dimer-dimer interface of each mutant, resulted in HIN T free energy estimates that were more accurate with respect to experimenta l data. To evaluate the ability of our method to predict free energies for de novo protein models, the same beta 37 mutants were computationally gener ated from native deoxy hemoglobin and similarly analyzed. Our theoretical m odels were sufficiently robust to accurately predict free energy changes in a localized region around the mutated residue. However, our method did not possess the capacity to generate the long-range secondary structural effec ts observed in crystallographically solved mutant structures. Final method analysis involved the computational generation of structurally and/or therm odynamically uncharacterized beta 37 deoxy hemoglobin mutants. HINT analysi s of these structures revealed that free energy predictions for dimer-tetra mer association in these models agreed well with previously observed energy predictions for structurally and thermodynamically characterized beta 37 d eoxy hemoglobin mutants.