The cytoskeleton functions as the central intracellular structure of organisms. It is composed of actin filaments, microtubules, and intermediate filaments, and many other proteins. Among the proteins that organize actin filaments are α-actinins, which form homodimers consisting of an actin-binding domain, a neck, a rod-shaped domain in the form of spectrin repeats, and a calmodulin-like domain that binds calcium ions. α-actinin-1 is one of the most important cross-linkers of actin in focal junctions and stress fibers, and its activity is thought to be regulated by calcium. The exact conformational changes upon calcium ion binding are still the subject of research, but a model of regulation of α-actinin-1 by calcium ions has recently been discovered. Upon Ca$^{2+}$ binding, α-actinin-1 is thought to be placed in a more closed conformation, thereby changing the way actin filaments are linked. To analyze the conformational changes upon calcium binding and the influence of α-actinin-1 on actin filament crosslinking, we prepared and expressed eight mutants, including four half-dimers and four wild-type proteins. We focused primarily on the interactions between the EF12 domain (with the calcium ion bound) and the rod domain that the mutations would cause. Three mutants of the wild-type protein contained a single mutation, one a double mutation; the same applies to mutants of the half-dimer (chains A and B), which contains all the functional domains of the complete wild-type α-actinin-1 protein.We performed site-specific mutagenesis by PCR, which was successful for all constructs. The mutants were then expressed in the host strain E. coli Bl21[DE3]pLysS, confirming that this bacterial strain is suitable for the expression of these mutants. Finally, we performed NaDS-PAGE to verify the amount of product in the soluble fraction and demonstrated that the α-actinin-1 mutants are predominantly expressed in the soluble form.
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