| Abstract
| - The computational modeling program HINT (Hydropathic INTeractions), an empirical hydropathic force field that includes hydrogen bonding, Coulombic, and hydrophobic terms, was used to modelthe free energy of dimer−tetramer association in a series of deoxy hemoglobin β37 double mutants. Fiveof the analyzed mutants (β37W → Y, β37W → A, β37W → G, β37W → E, and β37W → R) have beensolved crystallographically and characterized thermodynamically and subsequently made a good test setfor the calibration of our method as a tool for free energy prediction. Initial free energy estimates forthese mutants were conducted without the inclusion of crystallographically conserved water moleculesand systematically underestimated the experimentally calculated loss in free energy observed for eachmutant dimer−tetramer association. However, the inclusion of crystallographic waters, interacting at thedimer−dimer interface of each mutant, resulted in HINT free energy estimates that were more accuratewith respect to experimental data. To evaluate the ability of our method to predict free energies for denovo protein models, the same β37 mutants were computationally generated from native deoxy hemoglobinand similarly analyzed. Our theoretical models were sufficiently robust to accurately predict free energychanges in a localized region around the mutated residue. However, our method did not possess the capacityto generate the long-range secondary structural effects observed in crystallographically solved mutantstructures. Final method analysis involved the computational generation of structurally and/or thermodynamically uncharacterized β37 deoxy hemoglobin mutants. HINT analysis of these structures revealedthat free energy predictions for dimer−tetramer association in these models agreed well with previouslyobserved energy predictions for structurally and thermodynamically characterized β37 deoxy hemoglobinmutants.
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