Actin is one of the oldest and most common protein on Earth. Actin filaments play many important roles in the cell, especially when it comes to cell structure and movement. Many different proteins are responsible for the regulation and association of the actin network. One of them is α-actinin, a spectrin family crosslinker. It participates in cytoskeleton organization, cell division and movement and many other functions. It consists of an actin-binding domain, rod domain and a calmodulin-like domain, which in turn consists of EF12 and EF34 subdomains. There are four known human α-actinin proteins, of which two (ACTN2, ACTN3) are calcium insensitive and found in muscle, and two (ACTN1, ACTN4) are calcium sensitive and found in all cells. Calcium binding to ACTN1 reduces its ability to bundle actin filaments. The current prevailing theory is that the binding of a calcium ion to the EF12 subdomain stabilizes the protein structure and causes the EF34 subdomain to bind to the neck region, which changes the conformation of the actin-binding domain and reduces its ability to bundle actin filaments. To validate this theory, we have expressed a mutant with a GSGS linker inserted between the EF12 and EF34 subdomains. We determined the mutant′s affinity for calcium ions, actin bundling ability in the presence of calcium and thermostability both in the presence and absence of calcium. We then compared these characteristics to the wild type protein.
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