In this project, Dr. Josephs proposes to extend his extensive and elegant earlier work, that has culminated in a highly detailed model for the structure of the sickle cell hemoglobin fiber. This model provides the foundation for understanding the interactions that drive fiber formation and, thus, the clinical manifestations of sickle-cell anemia. The fundamental unit of the sickle-cell fiber is a double-strand of hemoglobin molecules that has been observed in crystals of deoxy hemoglobin molecules S (HbS). The fiber is composed of seven of these double strands that have a helical twist in the fiber, but not in the crystal. The crystal structure analysis by Love and colleagues delineates the interactions within the double strand, however the interactions between double strands that stabilize the fiber are not present in the crystal lattice. Thus, EM analysis, in conjunction with model building, is required to determine the "inter double strand" contacts. Two competing models have been proposed- one by the group of Dr. Josephs and the other by Edelstein and colleagues. With the use of an efficient microbial systems for production of recombinant hemoglobin, these alternate models can now be tested by site- directed mutagenesis. The proposed project contains three specific aims.