The broad, long range goal of this proposal is to elucidate the molecular basis of human red cell membrane destabilization resulting from hereditary hemolytic anemia related mutations. Ultimate achievement of this goal requires the development of a detailed understanding of the membrane skeleton's structural/functional/regulatory processes and its role in determining red cell stability in both normal and abnormal cells. The major immediate focus will be a detailed analysis of spectrin which is a major critical component of the red cell membrane. Normal spectrin and selected spectrin mutations will be studied in concert to attempt to better understand its role in stabilizing the red cell membrane. Three specific immediate goals are: 1) to determine the mechanism of spectrin self-assembly to form heterodimers and tetramers, 2) to locate the submolecular sites involved in self-assembly, and 3) to investigate the molecular shape of spectrin under different ionic conditions. Normal spectrin, as well as naturally occurring polymorphisms and pathogenic mutations, will be studied using established and novel approaches. Spectrin domain peptides defined by mild tryptic cleavage at 0 degrees C will continue to be important tools for these submolecular functional and structural analyses. Modem protein chemical methods will be used, together with: FPLC gel filtration based functional assays for spectrin assembly; completion of a 2D gel computerized database for spectrin domains; use of electron paramagnetic resonance (EPR) and fluorescence energy transfer analyses to measure intramolecular distances of spectrin in different environments; and expression of recombinant peptides to locate and characterize the minimum heterodimer nucleation site in the alpha subunit. These investigations should substantially contribute to at least five different conceptual aspects of spectrin that are currently poorly understood, but central to development of a comprehensive understanding of the red cell membrane. These critical questions are: 1) how are conformational changes resulting from mutations or protein-protein assembly transduced over long molecular distances in spectrin; 2) what is the mechanism of spectrin assembly and the factors that regulate assembly; 3) where are the specific sites of protein-protein interaction and how are they regulated; 4) what is the actual molecular shape of spectrin in the intact cell, - is it an elastic, compact 60 nm or an extended 200 nm tetrameric actin crossbridge; 5) what is the conformation of spectrin?
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