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 fusion proteins in Escherichia coli. Based on the evidence of circular dichroism spectra and cooperative thermal unfolding profiles both truncated 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 equilibrium 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 concentration-dependent association, with dissociation constants, estimated by sedimentation equilibrium, in the 1-10 microM range. Thus, in confirmation of earlier work, all four repeats are required to generate a maximally stable anti-parallel dimer (Kd approximately 10 pM), suggesting the presence of binding sites in all of them to allow for aligned pairing.