• Domain swapping, fragment complementation, and self-assembly in engineered chimeric proteins.

      Loh, Stewart; Craft, Matthew (2014)
      Domain swapping is a form of protein oligomerization in which two or more identical proteins reciprocally exchange parts of their tertiary structure. The structure of the domain swapped proteins is identical to the structure formed by the monomer except for the hinge region linking the two domains. Domain swapping provides the cell with a way to control complex assembly, alter enzyme kinetics and specificity. Domain swapping can also be used to reconstitute enzyme function or as a form of molecular recognition. Only a small number of proteins are known domain swap, and the forces behind domain swapping are not well understood. Much more need to be understood abouthow and why proteins domain swap, before it would be possible to reliably engineer proteins to do so. In an effort to understand the thermodynamic forces that drive domain swapping, the goal of this project was to induce domain swapping and investigate the effects different hinge regions have on an otherwise identical domain swapped structure. To accomplish this we inserted ubiquitin (Ub) into five surface loops of ribose binding protein (RBP), a protein that does not naturally domain swap. The presenceof ubiquitin puts conformational strain on RBP and vice versa, where the folding of one causes the other to unfold. The entropic penalty for having unfolded domains can be relieved by domain swapping, allowing all protein domains to be folded. Using gelfiltration and circular dichroism we determined that our RBP-Ub (RU) fusion proteins domain swap. This domain swapping is dependent upon the conformational strain caused by a folded Ub, and can be reversed by the addition of a flexible glycine linker. Using our RU system we provide the first evidence that proteins with non-identical hinge regions can domain swap to form stable, functional oligomers. Finally we present a physical model that explains the ability of different RU’s to domain swap, which provides at least some of the criteria required of domain swapping proteins.