Design of biologically active binary protein 2D materials

Abstract

Ordered two-dimensional arrays such as S-layers 1 , 2 and designed analogues 3 – 5 have intrigued bioengineers, 6 , 7 but with the exception of a single lattice formed with flexible linkers, 8 they are constituted from just one protein component. For modulating assembly dynamics and incorporating more complex functionality, materials composed of two components would have considerable advantages. 9 – 12 Here we describe a computational method to generate co-assembling binary layers by designing rigid interfaces between pairs of dihedral protein building-blocks, and use it to design a p6m lattice. The designed array components are soluble at mM concentrations, but when combined at nM concentrations, rapidly assemble into nearly crystalline micrometer-scale arrays nearly identical (based on TEM and SAXS) to the computational design model in vitro and in cells without the need for a two-dimensional support. Because the material is designed from the ground up, the components can be readily functionalized, and their symmetry reconfigured, enabling formation of ligand arrays with distinguishable surfaces which we demonstrate can drive extensive receptor clustering, downstream protein recruitment, and signaling. Using AFM on supported bilayers and quantitative microscopy on living cells, we show that arrays assembled on membranes have component stoichiometry and structure similar to arrays formed in vitro, and thus that our material can impose order onto fundamentally disordered substrates like cell membranes. In sharp contrast to previously characterized cell surface receptor binding assemblies such as antibodies and nanocages, which are rapidly endocytosed, we find that large arrays assembled at the cell surface suppress endocytosis in a tunable manner, with potential therapeutic relevance for extending receptor engagement and immune evasion. Our work paves the way towards a synthetic cell biology, where a new generation of multi-protein macroscale materials is designed to modulate cell responses and reshape synthetic and living systems.

Organizing Multi-Enzyme Systems into Programmable Materials for Biocatalysis

“…In addition, impressive progress has been made in the design and construction of self-assembling protein nanostructures that can form the foundation of new types of materials for biocatalysis [83,109,[130][131][132][133][134][135]. Recent publications demonstrate the design of diverse protein nanostructures including cages, layers, crystals, and filaments [136][137][138][139][140][141], the engineering of a preexisting cage into various structures [142][143][144][145], the assembly of computationally designed icosahedral nanostructures [137,138], 2D-arrays, crystals [146,147], and protein filaments [81], as well as the design of metal-coordination, disulfide bridges, and surface electrostatics for the creation of functional 2D-lattices and cages [146,[148][149][150][151][152].…”

The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

“…Maximum intensity projections were generated of the deskewed stacks. Correlative structured illumination and atomic force microscope: Correlative atomic force/fluorescence microscopy measurements were performed as described before (45). Atomic force microscopy (AFM) measurements were performed on a Bioscope Resolve AFM (Bruker), operated in PeakForce QNM mode, which was combined with a custom-built structured illumination microscopy system (46).…”

Fundamentals to function: Quantitative and scalable approaches for measuring protein stability

“…The importance of stability also extends beyond disease pathology. Stable proteins have industrial applications as catalysts for antibiotic and chemotherapeutic synthesis (Bruggink and Roy, 2001;Chen et al, 2006;Kondo and Hotta, 1999;Martin and Fischer, 1983;Tatsis et al, 2017;Volpato et al, 2010), catalysts for bioremediation and green chemistry (Peixoto et al, 2011;Sheldon and Woodley, 2018), lipases and proteases for detergents (Olsen and Falholt, 1998;Vojcic et al, 2015), flavoring and digestive additives (Merz et al, 2015;Raveendran et al, 2018), nanoengineering scaffolds (Ben-Sasson et al, 2021), and degradative enzymes for cellulosic biofuels (Reetz, 2013). To be used for these purposes, it is essential that native or engineered proteins not only adopt a particular fold but also remain folded under specific, and potentially harsh, conditions while still being able to carry out their desired functions.…”