Exploiting a rational β-strand insertion strategy and disulfide locking to mechanically manipulate domain-swapped protein structures

In domain swapping, the “hinge loop” region of the domain-swapped (DS) structure that connects two structurally arbitrary domains undergoes a large structural alteration. While previous studies have shed light on the role of the hinge region in changing the oligomerization state of proteins, our study highlights how the hinge loop region in a DS dimer protein can be meticulously manipulated to generate significantly altered protein structures without the need to change the oligomeric state of the protein. We illustrate how an odd versus even number of amino acid insertions in the hinge region alters the secondary structure by exploiting the basic principle of the beta strand “zigzag” conformation. These subtle changes result in predictable conformational alterations in the overall 3D structure of DS dimers while reducing the interdomain flexibility of the structure using a disulfide bond cross-linking strategy.