I propose to carry out X-ray crystallographic studies on two transition state analoques of haemoglobin. One cross-linked in the oxy or R structure so that it maintains this structure even when deoxygenated, the other cross-linked in the deoxy or T structure which should maintain this structure at low temperature, even when liganded. These analyses should be done at a resolution sufficient to refine the structures and obtain atomic coordinates, so as to be able to see the changes in conformation of the haems and the surrounding globin, and the possible transimission of such changes to the subunit boundaries. I also propose to work on species adaptation in the haemoglobin molecule. Biochemists and physiologists have discovered cooperative binding of oxygen to be the only property that is common to all vertebrate haemoglobins, while their response to other ligands is subject to wide variations, determined by the animals' environment and habits. By working out the stereochemical mechanism of several such adaptive mechanisms I discovered that responses to different chemical stimuli can evolve in a proteins by very few amino acid substitutions. I propose to contrinue this research. I plan to collaborate with Dr. Donald Abraham at the University of Pittsburgh on X-ray studies of the binding sites of potential anti-sickling drugs in the haemoglobin molecule, with the aim of improving the design of such drugs on a rational basis and thus raising their potency. I plan to do further X-ray studies of other abnormal haemoglobins in order to learn more about the stereochemical basis of congenital diseases in general, and also about the allosteric mechanism and the intramolecular forces in haemoglobin.
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