Designing Two-Dimensional Protein Arrays through Fusion of Multimers and Interface Mutations

Abstract

We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 μm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D.

A Self‐Assembling Two‐Dimensional Protein Array is a Versatile Platform for the Assembly of Multicomponent Nanostructures

“…TTM lattices routinely grow to tens of micrometers while retaining long‐range order, and hold great promise to control the 2D assembly of inorganic, synthetic, and proteinaceous components for several reasons. First, the initiation of TTM assembly strictly depends upon the addition of divalent metal cations whose identity and concentration control the kinetics and extent of polymerization . Second, the top and bottom faces of TTM lattices are nearly flat, as illustrated in Figure B, and demonstrated by AFM imaging elsewhere .…”

“…Following in the footsteps of Sleytr et al who showed that proteins from the S‐layers (surface layers) of certain archaea and bacteria could be reassembled into technologically useful crystalline 2D lattices, a number of groups have used protein engineering and protein design to create more flexible protomers that self‐assemble into robust 2D arrays . These approaches typically take advantage of rotational symmetry and rely on a variety of schemes to “stitch” the lattice together including the use of streptavidin, small ligands, metal coordination, disulfide bonds, coiled‐coil interactions, gene fusions with redesigned interfaces, and even subunits designed for 2D self‐assembly from scratch . To date, however, there has been little exploration of the potential of this new generation of 2D protein arrays for supramolecular assembly.…”

“…Previously, we used the Rosetta software environment to identify and computationally redesign STM4215, a Salmonella typhimurium homohexamer of unknown function for self‐assembly into thin (≈5 nm) hexagonal 2D arrays with ≈7 nm periodicity (Figure A) . The building block (Figure A) is called TTM (for Truncated‐Truncated‐Mutated) and consists of two truncated STM4215 monomers fused to one another through a hexaglycine linker.…”

“…The building block (Figure A) is called TTM (for Truncated‐Truncated‐Mutated) and consists of two truncated STM4215 monomers fused to one another through a hexaglycine linker. In the redesigned interface, multiple mutations eliminate steric hindrances and charge repulsions . TTM lattices routinely grow to tens of micrometers while retaining long‐range order, and hold great promise to control the 2D assembly of inorganic, synthetic, and proteinaceous components for several reasons.…”

“…First, the initiation of TTM assembly strictly depends upon the addition of divalent metal cations whose identity and concentration control the kinetics and extent of polymerization . Second, the top and bottom faces of TTM lattices are nearly flat, as illustrated in Figure B, and demonstrated by AFM imaging elsewhere . Such a lack of topography is ideal for organizing nanoparticles on 2D surfaces.…”