Probing the energetics of proteins through structural perturbation: sites of regulatory energy in human hemoglobin.

Abstract

The sites of energy transduction within the human Hb molecule for the regulation of O2 affinity were determined by studying the molecule''s energetic response to structural alteration at individual amino acid residues. For 22 mutant and chemically modified Hb the total free energy used by the tetrameric molecule for alteration of O2 affinity at the 4 binding steps was determined. The regulation of O2 binding affinity is apparently due to energy changes which are mostly localized at the .alpha.1.beta.2 interface. A high degree of internal cooperativity within this contact region is indicated; the structural perturbations at individual residue sites are energetically coupled. Cooperativity in ligand binding is a reflection of cooperativity at a deeper level, that of the protein-protein interactions within the .alpha.1.beta.2 interfacial domain.