Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials have potential applications in sensing, catalysis, and medicine. Whereas numerous designs have been implemented for the bottom-up construction of protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and, thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. The site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small molecule- and protein-functionalized surfaces with no loss of crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces toward the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

Automated summary

This study demonstrates the selective modification of designed two-dimensional protein crystals using phosphopantetheinyl transferases (PPTases), allowing for enzymatic functionalization on protein surfaces. The incorporation of enzymes into the protein arrays through covalent tethering of short peptides or molecular tags enables site-specific modification without affecting the crystalline order of the arrays. The work underscores the potential for chemoenzymatic modification of large protein surfaces to create sophisticated protein platforms resembling complex cell surfaces.