The broad, long range goal of this project is to develop a detailed understanding of the molecular properties of spectrin and its role in red cell membrane deformability and stability.
The specific aims are to: 1) explore structural and functional implications of our recent, exciting discovery that lysines in the spectrin tetramer binding site are selectively, extensively carbamylated in vivo; 2) determine the submolecular basis of spectrin's unique flexibility/extensibility properties and rationalize these properties with existing high resolution structures of spectrin motifs; 3) define the structure of the red cell spectrin tetramer binding site and investigate the isoform specificity of this site; and 4) determine the spectrin isoform specificity of the dimerization initiation site. Three major hypotheses will be tested, which should result in a more accurate, detailed understanding of red cell membranes as well as membrane skeletons of other cell types. The first hypothesis is that physiological modification of specific spectrin lysines in the red cell affect its function and could play a critical role in red cell survival under both normal and pathological conditions possibly including renal failure, diabetes, and red cell aging. The second hypothesis is that red cell spectrin, which is more flexible than non-red cell isoforms, has a different conformation in solution than suggested by available high-resolution structures with long continuous helices connecting adjacent homologous motifs. The third hypothesis is that different spectrin isoforms share similar mechanisms of self-assembly but isoform-specific complementary recognition sites in the dimer initiation and tetramer binding regions control correct isoform assembly and determine association affinities. These hypotheses will be tested using isolated spectrin dimers and monomers, as well as recombinant domains of red cell and non-red cell spectrin isoforms. Functional properties including interactions between adjacent motifs, dimerization, and tetramer assembly will be studied using biochemical and biophysical techniques including isothermal titration calorimetry, analytical ultracentrifugation, and HPLC gel filtration. Structural properties will be analyzed using protein microchemistry methods that will include high-resolution 2D gels, in-gel protease digestion, mass spectrometry (MS), hydrogen-deuterium exchange/MS analyses, and related methods.
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