The goal of this work is to study the structure and assembly of polymers of sickle cell hemoglobin which are responsible for sickle cell anemia. These studies will involve electron microscopy of polymers in various stages of assembly. Rapid freezing in liquid ethane will be used to arrest the assembly process and the partially assembled polymers will be examined in the electron microscope. In a similar vein, studies of polymer disassembly will also be carried out in order to determine how this process occurs. The interpretation of these studies will draw heavily from existing models of the heterogeneous polymerization of HbS. A prime objective of the work is to identify the physical structures which mediate the association and dissociation reactions. Studies of the molecular structure of the fiber have been carried as far as seems possible using negative staining. We propose to extend these studies to higher resolution using cryomicroscopy. This technique involves rapidly freezing specimens in a thin layer of vitreous ice. Micrographs record the electron density of the specimen. This technique will allow us to extend the resolution of micrographs of HbS polymers from the existing 32A limit to 15A or better. This increase in resolution will provide a far more detailed image of the molecular structure of sickle hemoglobin polymers than has been possible to obtain up to now. These higher resolution data, taken in conjunction with the known atomic structure of HbS should permit mapping many of the intermolecular contacts which stabilize these clinically important structures.
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