The goal of this research program is to determine the molecular mechanism of cooperative oxygenation in human hemoglobin. Dr. Ackers is attempting to decipher the rules and energetic driving forces whereby the ten molecular forms (each with a structurally-unique combination of ligated and unligatd heme sites) respond to the interactions with heme-site lignads and small regulatory molecules including protons, chloride ions, and 2,3-diphosphoglycerate. A breakthrough was recently achieved in this problem when methods were developed that resolve the functional energetics of hemoglobin tetramers in all ten states of hemesite ligation, using non-oxygen ligands. This has opened the door to obtaining previously inaccessible knowledge regarding properties of the intermediate species and their roles in the cooperative mechanism. In current work studies will be extended to mutant hemoglobins in order to map the structural pathways of free energy transduction and to establish the connections with oxygen binding. This research will use a combination of techniques, including quantitative low temperature isoelectric focusing, and high precision binding of oxygen, protons, and chloride as a function of temperature and organic phosphates. The binding measurements will be correlated with circular dichroic studies of quaternary structure. The results from this research will serve as a model for studying "molecular codes" in other complex regulatory protein assemblies.
