Engineering covalent loops in proteins can serve as an on/off switch to regulate threaded topologies.

Knots in proteins are under active investigation motivating refinements of current techniques and the development of tools to better understand the knotted topology. A strong focus is to identify new knots and expand upon the current understanding of their complex topology. Previous work has shown that the knotted topology, even in the simplest case of knots, encompasses a variety of unique challenges in folding and tying a chain. To bypass many of the in vitro experimental complications involved in working with knots, it is useful to apply methodologies to a more simplified system. The pierced lasso bundles (PLB), we discovered where a single disulphide bridge holds the threaded topology together, presents a simpler system to study knots in vitro. Having a disulphide bridge as an on/off switch between the threaded/unthreaded topology is advantageous because a covalent loop allows manipulation of the knot without directly altering affecting secondary and tertiary structure. Because disulphide bridges are commonly used in protein engineering, a pierced lasso (PL) topology can be easily introduced into a protein of interest to form a knotted topology within a given secondary structure. It is also important to take into account that if formed, disulphides can inadvertently introduce an unwanted PL. This was found upon determination of the crystal structure (PDB code 2YHG) of the recently de novo designed nucleoside hydrolase. Our detailed investigations of the PL presented here will allow researchers to look at the introduction of disulphide bridges in a larger context with respect to potential geometrical consequences on the structure and functional properties of proteins.