The broad goal of our research is to understand the basic processes involved in control and regulation through allosteric macromolecules. The respiratory proteins are particularly well suited by the nature of their physical properties, diversity and availability. They exhibit a wide range of aggregation states, ranging from monomeric forms to large multi-subunit aggregates. Their reactions with gaseous ligands, such as oxygen and carbon monoxide, can be explored with unparalleled precision due to spectral changes upon ligation and to the ability to produce or measure accurate ligand activities in terms of their partial pressures. They form macromolecular systems that can change conformation or aggregation states upon ligand binding. Natural and constructed mutants as well as materials from various animal species provide a range of systems for exploring the structural basis of the functional properties of cooperativity and ligand regulation.
The specific aims of this project are characterization of homotropic and heterotropic linkage effects of allosteric proteins (hemoglobin and hemocyanin), ligand regulated aggregation and phase formation , and development of a thermodynamic framework for analysis of generalized ligand binding. Determination of precise gaseous ligand binding curves as a function of auxiliary ligand conditions provides key thermodynamic information and data required for molecular model description of the underlying reaction processes. Thin layer optical, optical titration cells, and microcalorimetric methods have been developed especially for these studies. Global data analysis of multiple experiments, computer simulation of model situations, and formulation of suitable molecular models provide the theoretical basis for understanding the quantitative properties of macromolecular ligand control. The special problems to be addressed are: 1) Determination of equilibrium gas ligand binding curves using multiple wave length measurements in UV, visible, and near IR by thin layer optical cell and special optical titration method; 2) Comparative binding curve measurements using scanning reaction cell with oxygen electrode (Imai technique); 3) Evaluation of non-linear optical absorption with degree of ligation; 4) Measurement of heat binding curves at several temperatures and evaluation of free energy, enthalpy, entropy changes for oxygen and carbon monoxide binding; 5) Study of phosphine (P(CH3)4) ligation to hemoglobin by NMR and optical spectroscopy; 6) Determination of oxygen binding properties in hemoglobin crystals by titration and x-ray methods; 7) Study of ligand linked aggregation properties of bird hemoglobins by thin layer methods and use of light scattering molecular weight determinations.
Showing the most recent 10 out of 44 publications
