Self-Association of Spectrin's Repeating Segments

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Abstract
We have examined the self-association behavior in solution of one of the repeating conformational segments of Drosophila spectrin, D-α-14, as well as of the two-segment unit, D-α-14,15. In both polypeptides, sedimentation equilibrium and nondenaturing gel electrophoresis detect a reversible, moderate affinity (K2 ≅ 104 M-1) dimerization reaction. Equilibration between monomer and dimer is kinetically limited near 5 °C, but occurs at a measurable rate at temperatures ≥20 °C. The temperature dependence for equilibration is consistent with the requirement for extensive disruption of helix−helix packing as the reaction proceeds in either direction. Hydrodynamic studies by means of sedimentation velocity confirm that in solution the C helix in the monomer of D-α-14 is folded back to interact with the A and B helices, and that the form of the monomeric subunit observed in the crystal structure, in which the B and C helices are continuous, does not persist in the monomer in solution. Both the dimer of D-α-14 and the monomer of D-α-14,15 appear to be twice the length of the D-α-14 monomer, while the frictional ratio of the D-α-14,15 dimer is consistent with four end-to-end triple α-helical domains.