HYDROPATHIC ANALYSIS OF THE NONCOVALENT INTERACTIONS BETWEEN MOLECULAR SUBUNITS OF STRUCTURALLY CHARACTERIZED HEMOGLOBINS

The software program, HINT (Hydropathic INTeractions), which character izes non-polar-non-polar, polar-polar, and non-polar-polar interaction s, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. H INT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters e xperimentally determined from solvent transfer experiments. The main f ocus of this study is to compare HINT scores that are based upon exper imentally and thermodynamically derived measurements with experimental ly determined thermodynamic results. The HINT analysis yields a good f irst-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembl y for mutant hemoglobins. The results provide a framework for understa nding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the alpha 1 beta 1 and alpha 2 beta 2 subunit contacts are stabil ized via several polar and many hydrophobic interactions with few repu lsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglob in) structures; (2) the alpha 1 alpha 2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the alpha 1 beta 2 and alpha 2 beta 1 contacts have both strong po sitive and negative interactions in both T and R states with few hydro phobic interactions. The HINT scoring methodology provides a quantitat ive characterization of the major role of the alpha 1 beta 2 and alpha 2 beta 1 interfaces in the T --> R quaternary transition. HINT also c onfirms the stronger hydrogen bond formation in mutant Kb Rothschild ( Trp 37 beta --> Arg) with Asp94 alpha 1 that gives rise to a low-affin ity (deoxy) hemoglobin. HINT shows that the stabilization of the alpha 1 beta 2 interface with mutant Hb Ypsi-lanti (Asp99 alpha --> Tyr) pr oduces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specifi c sites for mutagenesis at the alpha 1 beta 2 interface that can be ex plored to shift the allosteric equilibrium in either direction. In add ition, the HINT program provides useful diagnostic data for checking t he quality of refined crystallographic structures. (C) 1997 Academic P ress Limited.