The goal of the proposed research is to investigate the structure-function relationship under physiological conditions at atomic resolution of allosteric proteins and enzymes using hemoglobin (Hb) as a model. There are several features inherent in Hb, which make this molecule a unique choice for gaining insights into the signal pathway for information transfer from on subunit to another at the atomic level. In order to gain a new understanding regrading the structure and properties of Hb which allow this molecule to serve as an oxygen carrier in vertebrates, we plan to apply techniques of biochemistry, biophysics, molecular biology, and structural biology to our Hb research and to correlate the results obtained from nuclear magnetic resonance (NMR) spectroscopy, x-ray crystallography, optical spectroscopy, computer modeling, and equilibrium and kinetic studies of the binding of oxygen and other ligands to Hb. Even though Hb is one of the best studies proteins, many details are not fully understood and some aspects are controversial. For example, there are at least three quaternary structures (T,R, and R2) of Hb in crystals and the functional properties of Hb in crystals are distinctly different from those in solution. There is no information on the solution structure of Hb. In order to correlate the structure and function of Hb under physiological conditions and to resolve the conflicting results derived from studies obtained from Hb crystals and Hb in solutions, we need to know the details of the structures and dynamics of Hb as a function of oxygenation in solution. We propose to carry out such a study using multinuclear, multidimensional NMR spectroscopy. In addition, we also propose to continue our research in the areas of sickle cell anemia and Hb-based oxygen carriers. Hb research is an excellent illustration of how the discoveries from basic research on proteins can make important contributions to medicine and biotechnology. Some of the proposed experiments will be carried out in collaboration with experts who have appropriate facilities not available in our laboratory.
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