FURTHER ANALYSIS OF THE ROLE OF SPECTRIN REPEAT MOTIFS IN ALPHA-ACTININ DIMER FORMATION

Protein constructs consisting of repeats 1-4, repeats 1-3 and repeats 2-4 of the rod domain of chicken alpha-actinin were expressed as fusio n proteins in Escherichia coli. Based on the evidence of circular dich roism spectra and cooperative thermal unfolding profiles both truncate d rod fragments were judged to have assumed the native structural fold . The thermal stabilities were in both cases significantly lower than that of the intact rod (repeats 1-4). Analyses by sedimentation equili brium and velocity provided further evidence to show that fragment 1-4 is entirely dimeric in the concentration range of these experiments, resembling therefore the rod domain isolated by proteolytic digestion of native alpha-actinin. Fragment 2-4, and probably also 1-3, show con centration-dependent association, with dissociation constants, estimat ed by sedimentation equilibrium, in the 1-10 mu M range. Thus, in conf irmation of earlier work, all four repeats are required to generate a maximally stable anti-parallel dimer (K-d similar to 10 pM), suggestin g the presence of binding sites in all of them to allow for aligned pa iring.