The broad, long range goal of this project is to eluciate the mechanisms of human red cell spectrin sef-assembly and the molecular bases for perturbing functional interactions by hereditary hemolytic anemia related mutations. In the current proposal, three working hypotheses will be explored. The first hypothesis is that phosphorylation of beta spectrin influences membrane stability by affecting the rate limiting step in tetramer formation. The second hypothesis is that alpha spectrin pathogenetic mutations located substantial molecular distances away from the tetramer binding site affect tetramer formation by affecting the closed yields open dimer equilibrium. A third hypothesis is that current spectrin models deduced from the crystal structure of a single motif where adjacent motifs are connected by a continuous helix are inaccurate; instead, we propose that spectrin's flexibility is maintained by in-register alignment of laterally paired alpha and beta motifs with flexible hinge regions connecting adjacent motifs. The specific goals of the current proposal are: 1) to identify the beta spectrin phosphorylation sites and determine their function; 2) to determine whether the rate limiting closed yields open dimer equilibrium is affected by pathogenic mutations in the hairpin loop region; and 3)to determine how inter-motif and lateral interchain non- covalent interactions affect conformational stability. These specific goals will be pursuedusing molecular biology and protein chemical methods. Most experiments will utilize normal and mutagenized recombinant spectrin peptides, intact spectrin monomers, and in vivo phosphorylated spectrin dimers. Spectrin phosphorylation, assembly, and structure will be studied using: HPLC peptide mapping, mass spectrometry, protein microsequencing, protein binding assays, microcalorimetry, and related methods. The four specific aims are: 1) Structural and functional role phosphorylation in spectrin assembly; 2) Role of the closed hairpin loop in spectrin function; 3) Mechanism of membrane destabilization by pathogenic spectrin mutations locaed at lare molecular distances from the tetramerization site; 4) Analysis of lateral dimer interfaces and inter-motif interfaces and their effects on conformational stability of spectrin.
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