Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: Systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generati on, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse p opulation of its mutants has been thermodynamically characterized. Noncoval ent interactions for all computationally generated hemoglobin mutants are q uantitatively examined with the molecular modeling program HINT (Hydropathi c INTeractions), HINT scores all biomolecular noncovalent interactions, inc luding hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, bas e-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" t hat are translated into free-energy estimates. Analysis of 23 hemoglobin mu tants, in both deoxy and oxy states, indicates that the effects of mutant r esidues on structurally bound waters (and visa versa) are important for gen erating accurate free-energy estimates. For several mutants, the addition/e limination of structural waters is key to understanding the thermodynamic c onsequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78 , standard error = 1.4 kcal mol(-1), n = 23), Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, stan dard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three o utliers from this data set results in a better correlation of r = 0.87 (slo pe = 0.72, standard error = 0.75, n = 20). These three mutations may signif icantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifi cally, we find an acidic residue within the native deoxy hemoglobin dimer-d imer interface that may be protonated at physiological pH. The final analys is is a model design of novel hemoglobin mutants that modify cooperative fr ee energy (DeltaG(c))-the energy barrier between the allosteric transition from deoxy to oxy hemoglobin, Proteins 2001;42: 355-377. (C) 2000 Wiley-Lis s, Inc.