The long-term objective of this proposal is to understand the molecular basis of flexibility of the ubiquitous cytoskeletal protein, spectrin, and its relatives, alpha-actinin and dystrophin. Continuing the previously successful strategy of determining the X-ray crystal structure of two connected repeating units of chicken brain alpha-spectrin, which led to the proposal of two of the first molecular models of spectrin flexibility, a follow-up investigation is proposed to critically test those models. The strengths of X-ray crystallography, fluorescence and nuclear magnetic resonance (NMR) spectroscopy will be exploited to address the following important questions about the models: 1) Is the conformation of a linker region coordinated with that of an adjacent linker region in a three repeat fragment? 2) Are linker regions predicted to be a random coil by secondary structure prediction methods nonhelical (which, if true, could suggest yet a third model of spectrin flexibility and also offer the possibility of studying mutations correlated with hereditary elliptocytosis)? 3) How does the absence of the nearly invariant tryptophan affect the conformation of the linker region and flexibility of two connected repeats? 4) Is conformational rearrangement of one of the previously determined structures-a key feature of one of the models--due to the phasing or to the sequence of the construct? To answer these crucial questions concerning models of spectrin flexibility, 3 structures will be determined by X-ray crystallography, 2 will be studied by fluorescence energy transfer and 10 will be analyzed by NMR. These three approaches will complement each other as the X-ray crystal structures will provide atomic distances for interpretation of energy transfer data and vector orientations for interpretation of NMR data, and energy transfer and NMR data will provide dynamic information about the crystal structures. The cloned spectrin fragments will also be characterized by their circular dichroism and fluorescence spectra, by their stabilities to urea and thermal denaturation and by their molecular weights on analytical ultracentrifugation. Proposed critical testing of molecular models of spectrin flexibility will contribute fundamental knowledge likely to advance understanding of conditions such as hereditary elliptocytosis and spherocytosis, muscular dystrophy, hydrops fetalis and Fanconi anemia, in all of which spectrin or spectrin-related proteins are abnormal or reduced in amount.
