Our long-term objective is to understand the molecular basis of human erythrocyte shape transformation. Because the mechanochemical properties of the erythrocyte membrane and its spectrin-based membrane skeleton are key determinants of shape and stability, the specific objectives for this grant period are to gain a molecular understanding of the binding properties, chain dynamics, and structure of spectrin. DNA constructs will be used to express individual native spectrin chains, fragments of these chains, and genetically altered spectrin chains or fragments in E. coli and other expression systems. These spectrin chains or fragments will be used in reconstitution assays to analyze the precise location, primary structure, and biochemical characteristics of spectrin binding sites that are known to be essential in spectrin function and erythrocyte shape and stability. In collaboration with a polymer physics laboratory, the spectrin chains or fragments will also be used to obtain a mechanical description of the forces associated with the deformation of spectrin molecules at the length scales of 5 nm and higher. For these experiments, the dynamics of native spectrin chains as well as genetically altered chains will be analyzed using transient electrical birefringence and new resonator devices designed for measuring the hydrodynamic properties of biological polymers that are available only in milligram quantities. In other collaborations, the atomic structure of spectrin's repeating motifs will be determined by X-ray diffraction and by NMR. A spectrin deficient erythroleukemic cell line will be developed to provide an assay system in which some of the in vivo consequences of alterations in spectrin binding activities or chain dynamics can be assessed for their effects on some of spectrin's well known functions, such as membrane stabilization, domain segregation, and shape.
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