This proposal is aimed at studying the molecular events involved in sickle cell hemoglobin polymerization and the consequent red cell deformation resulting from it. The ultimate goal of the project is to apply the information obtained from the studies to the amelioration of painful crises in sickle cell anemia patients. Beyond this practical application, the studies should offer insight into other systems involving macromolecular assemblies. Hb S polymerization proceeds by two distinct mechanisms: the first is a nucleation event leading to initial polymerization, and the second is an exponential increase in the rate of fiber formation as newly created surfaces nucleate additional fibers. Although the molecular contacts within fibers are known, little or nothing is known about the interfiber contacts or of the mechanisms involved in aggregation of fibers. Fifty- five mutants have been prepared so far and 33 affect fiber formation. Thirty-one occur at calculated intermolecular sites, and radial and linear growth are different. Thus, crystal coordinates by themselves have not allowed a full description of fiber propagation. Still less is known about the nature and formation of """"""""irreversibly sickled cells"""""""", although their existence has been known for more than 20 years. These cells are rigid and retain a sickled shape, even in the absence of fibers. How the cytoskeleton of the cell has been altered to produce this peculiar situation is not known, but perhaps the sensitivity of skeletal proteins (especially spectrin) to ionic conditions may provide a clue. This proposal has two overall goals: study of the structure of fiber bundles by negative staining and cryoelectron microscopy, and the preparation of site- directed hemoglobin mutants to explore how specific substitutions affect fiber formation. The mutants will be studied by electron microscopy and the polymerization kinetics will be evaluated by video-enhanced light microscopy. These molecular events will be correlated with cellular alterations using cryomicroscopy to avoid perturbations of the skeletal structures caused by staining. The proposal involves four specific aims: 1. The mechanism of polymerization. Fiber bundles will be studied using negative staining cryoelectron microscopy in order to determine intermolecular contacts that facilitate nucleation of new fibers on the surface of existing fibers. 2. Site directed mutagenesis. Double mutants of hemoglobin S will be prepared to determine the effect of specific alterations on fiber formation. 3. Studies of fibers in high concentrations of phosphate buffer. This section of the work will attempt to determine the physiological relevance of previous data collected in high phosphate. 4. The role of the red cell cytoskeleton in cell deformation. Cryomicroscopy will be used to study the fine structure of the cytoskeleton of irreversibly sickled cells.
Showing the most recent 10 out of 27 publications
