The general goals of this project are to establish solid structure- function relationships in ligand binding to myoglobin that include definitions of the roles of key amino acid residues, the roles of dynamics and conformational substates, and the role of water. Because of the accessibility of myoglobin to many spectroscopic techniques and the plethora of studies on this molecule, a unique opportunity exists to develop a complete biophysical understanding of the detailed ligand binding pathways. Using X-ray crystallography to determine three- dimensional structures and using computational tools such as electrostatic potential calculations and molecular dynamics simulations based on these structures, correlates between structure and function will be developed in a way not possible with other systems. In particular, the aims are to: 1) Determine structures of mutants designed to test ideas about the physiological function of myoglobin. 2) dynamic details of ligand binding pathways in mutants using cryo- crystallography. 3) Develop conformational substate models of key myoglobin complexes 4) Use electrostatic potential calculations to probe the role of charge and polarity on ligand orientation and binding 5) Use molecular dynamics simulations to calculate and then predict binding energies of ligands 6) Determine the crystal structures of chemically modified myoglobins to test specific theories on the role of metal movements in ligand binding and to answer other questions about the plasticity of the distal pocket. 7) Resolve questions about the details of CO binding in crystals versus sol ion 8) Determine the structures of mutants designed to test ideas about the folding and stability of the protein. The insight gained in the study of myoglobin also has a direct medical application. In the development of hemoglobin for a cell-free blood substitute, there are two factors where the results can and will be applied. First, the oxygen affinity must be re-tuned for a cell-free hemoglobin, and this can be achieved with mutations at the distal histidine position. Secondly, the protein must keep its heme tightly bound and reduced within the globin to be functional, and these studies on heme loss, mechanisms of oxidation and globin stability will help in the design process. Thus, the last specific aim is to: 9) Determine the structures of mutant hemoglobins and determine the extent to which myoglobin can serve as a simple model system for the development of blood substitute proteins. This grant application represents the structural components of a collaboration with Prof. John Olson and members of his laboratory, where the mutants are constructed, and their kinetics and other spectroscopic properties are measured. The two groups work jointly to design the mutants, to train students and postdoctoral trainees in all techniques used in both laboratories, and to synthesize meaningful structure-function correlates.
Showing the most recent 10 out of 34 publications
