The long-term objective of this work is to show how the function of the ubiquitous cytoskeletal protein, spectrin, follows from its unique molecular structure, over 90% of which consists of chains of 20 and 16 repeating units. These linked repeats not only possess a remarkable flexibility, but some of them have evolved to bind to the adaptor protein ankyrin, itself the key link between the cell membrane and the spectrin cytoskeleton. By this means, spectrin promotes cell membrane-cytoplasmic communication in nonerythroid cells and reversible deformation of erythroid cells, in which it is the major part of their flexible networks. The health relatedness of the molecular attributes of the spectrin-ankyrin interaction, reflecting the versatility of the spectrin repeat, is clear from the correlation between perturbation of the spectrin-ankyrin binding site and nearby regions with a variety of hemolytic anemias, with intra-erythroid maturation of malaria parasites and with a subset of cardiac arrhythmias. Spectrin repeat-like domains have even been found recently in the amyloid precursor protein forming plaques leading to Alzheimer's disease. To further understanding of these pathologies, regions of mutually interacting spectrin and ankyrin will be cloned in bacteria and purified to study their properties. A three-part study of the ankyrin binding domain of spectrin and the spectrin binding domain of ankyrin will include 1) measurements of their circular dichroism, fluorescence, thermodynamic stabilities of folding and molecular size in solution, 2) measurement of their binding by GST pull-down assays, by isothermal titration calorimetry and by analytical ultracentrifugation to equilibrium and 3) determination of the molecular structures of these proteins singly and together by X-ray crystallography. This approach, which led to the first molecular models of flexibility of linked repeats of spectrin, will indicate how the ankyrin binding repeats of spectrin achieve specific and high affinity interaction with the spectrin binding site of ankyrin and how mutations and other perturbations can undermine this pivotal process.
