α-actinin is an actin-binding protein which plays an important role in many cell structures, from the contractile ring to the cytoskeleton and sarcomeres, and is also involved in cell motility. It forms a dimer, both units of which are composed of an actin-binding domain, neck region, spectrin repeats and calmodulin-like domain. Calcium sensitive and insensitive forms are known to exist. In this work we wanted to identify where the most important conformational changes happen upon calcium binding to the calcium sensitive α-actinin 1. For this purpose we have – based upon the crystal structure of α-actinin 1 bound to Ca2+ – constructed, cloned and expressed three mutants, each of which locks part of the structure of α-actinin into the calcium-bound form. We have then purified the mutants using a variety of chromatographic techniques, and, for the mutants in which we have introduced cysteine residues on different chains – which should lead to the formation of covalently bound dimers – checked for covalent dimer formation on SDS-PAGE. We have confirmed the formation of dimers by one of the the two mutants in which we expected such formation to occur. We have tried to encourage the formation of dimers in the other mutant by incubation in an oxidizing environment and in the presence of calcium, but were unsuccessful. The mutants will be used for analysis of formation of actin bundles.
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