De novo design of autocatalytically forming intra- and intermolecular isopeptide bonds to construct rigid covalent protein assemblies

In a groundbreaking study, researchers have designed entirely new proteins capable of forming intramolecular and intermolecular isopeptide bonds in an autocatalytic manner. These unique bonds are akin to the pili found in Gram-positive bacteria. The team successfully devised over 50 distinct designs, which were subsequently confirmed through mass spectrometry and crystal structures.

These innovative isopeptide bonds were then adapted to create split proteins. When these split proteins were combined, they formed a covalent intermolecular isopeptide crosslink. Crucially, this crosslinking process is orthogonal to the existing SpyTag/Catcher system, meaning the two can operate independently. Furthermore, the formation of these crosslinks can be controlled by altering the temperature, offering precise regulation over the timing of crosslinking in protein assemblies.

Building upon this foundation, the researchers extended their designs to construct rigid domain crosslinks. This enabled the creation of large, well-ordered symmetric rings, with some rings reaching up to 215 kDa in size. Remarkably, these rings were irreversibly covalently crosslinked by multiple isopeptide bonds, resulting in a single, cohesive molecule.

This discovery not only sheds light on the factors that govern isopeptide bond formation but also dramatically expands the repertoire of isopeptide bond crosslinking systems.

The researchers' findings provide a comprehensive framework for constructing fully covalent rigid protein assemblies. This advancement opens up new possibilities for the manipulation and engineering of protein structures at an unprecedented level of precision and control.