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

Abstract

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.

Protein Assembly by Design

“…Two different designed, selfassembled protein arrays (2D Zn-mediated RIDC3 crystals and disulfide-linked C98 RhuA lattices), whose surfaces can be tagged with functional sites (short peptide ybbR or molecular tag CoA), were prepared for enzymatic modification by Sfp PPTase (Figure 81a). 594 Notably, the site-specific modification of both 2D arrays could be carried out genetically or chemically without disrupting the underlying crystal lattice packing, characterized by TEM images of the protein crystals and confocal microscopy images of the labeled tags. This study highlights the potential for chemoenzymatic modification of 2D protein arrays toward the hierarchical construction of multicomponent protein systems.…”

“…To generate functionalized 2D protein materials from the bottom up, Subramanian et al developed an enzyme-directed surface modification approach to site-selectively tailor the surface of 2D protein crystals by using Sfp phosphopantetheinyl transferase (PPTase). Two different designed, self-assembled protein arrays (2D Zn-mediated RIDC3 crystals and disulfide-linked C98 RhuA lattices), whose surfaces can be tagged with functional sites (short peptide ybbR or molecular tag CoA), were prepared for enzymatic modification by Sfp PPTase (Figure a) . Notably, the site-specific modification of both 2D arrays could be carried out genetically or chemically without disrupting the underlying crystal lattice packing, characterized by TEM images of the protein crystals and confocal microscopy images of the labeled tags.…”

“…Within the nanosheets, energy absorbed by donors can be transferred to acceptors by direct FRET or successive donor-to-donor transfers, conferring light harvesting properties to the nanosheets. (a) Adapted with permission from ref . Copyright 2020 ACS.…”

Design of a Hierarchical Assembly at a Solid–Liquid Interface Using an Asymmetric Protein Needle

“…Proteins can hierarchically assemble into well-defined supramolecular structures. Oligomeric assembly units can be formed with a defined number of polypeptides, which can further lead to extended assembly structures, such as fibers, sheets, and crystals. − Among these structures, two-dimensional assemblies are attractive candidates for development of bio-nanomaterials because their periodicity and bioactivity are useful properties for molecular displays, optics, and sensors. − In efforts to mimic natural two-dimensional assemblies, many researchers have artificially constructed crystalline protein assemblies. − However, the construction of such artificial assemblies requires intensive design efforts, which consider the complexity of protein–protein interactions. Previous investigations have overcome these obstacles by exploiting structural symmetry to reduce the number of protein–protein interfaces under consideration. , Consequently, conventional artificial protein assemblies must be designed with atomic-level accuracy to avoid unexpected interactions.…”

Controlled and Stable Patterning of Diverse Inorganic Nanocrystals on Crystalline Two-Dimensional Protein Arrays

“…Protein assemblies are promising scaffolds for the development of hierarchical nanomaterials as they can be engineered with angstrom precision and self-assemble into stable, complex architectures. ,− Covalent Au–thiol bonds have been successfully exploited to target AuNPs to Cys side chains, ,,, but the intrinsic reactivity and structure encoded within protein assemblies should allow for varied and orthogonal targeting strategies. , This richness of protein biochemistry has yet to be exploited to create materials with close and well-defined interactions between different classes of nanoparticles.…”