Protein switches perform essential roles in many biological processes and are exciting targets for de novo protein design, which aims to produce proteins of arbitrary shape and functionality. However, the biophysical requirements for switch function - multiple conformational states, fine-tuned energetics, and stimuli-responsiveness - pose a formidable challenge for design by computation (or intuition). A variety of methods have been developed toward tackling this challenge, usually taking inspiration from the wealth of sequence and structural information available for naturally occurring protein switches. More recently, modular switches have been designed computationally, and new methods have emerged for sampling unexplored structure space, providing promising new avenues toward the generation of purpose-built switches and de novo signaling systems for cellular engineering.

Self-assembly of molecular building blocks into higher-order structures is exploited in living systems to create functional complexity and represents a powerful strategy for constructing new materials. As nanoscale building blocks, proteins offer unique advantages, including monodispersity and atomically tunable interactions. Yet, control of protein self-assembly has been limited compared to inorganic or polymeric nanoparticles, which lack such attributes. Here, we report modular self-assembly of an engineered protein into four physicochemically distinct, precisely patterned 2D crystals via control of four classes of interactions spanning Ångström to several-nanometer length scales. We relate the resulting structures to the underlying free-energy landscape by combining in-situ atomic force microscopy observations of assembly with thermodynamic analyses of protein-protein and -surface interactions. Our results demonstrate rich phase behavior obtainable from a single, highly patchy protein when interactions acting over multiple length scales are exploited and predict unusual bulk-scale properties for protein-based materials that ensue from such control.

Many proteins exist naturally as symmetrical homooligomers or homopolymers¹. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design^2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures^1,6-a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, 'one-pot' coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe³⁺ and Zn²⁺ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures^7,8 and are, to our knowledge, unique among designed systems^9-13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design.

The phyllosilicate mineral muscovite mica is widely used as a surface template for the patterning of macromolecules, yet a molecular understanding of its surface chemistry under varying solution conditions, required to predict and control the self-assembly of adsorbed species, is lacking. We utilize all-atom molecular dynamics simulations in conjunction with an electrostatic analysis based in local molecular field theory that affords a clean separation of long-range and short-range electrostatics. Using water polarization response as a measure of the electric fields that arise from patterned, surface-bound ions that direct the adsorption of charged macromolecules, we apply a Landau theory of forces induced by asymmetrically polarized surfaces to compute protein-surface interactions for two muscovite-binding proteins (DHR10-mica6 and C98RhuA). Comparison of the pressure between surface and protein in high-concentration KCl and NaCl aqueous solutions reveals ion-specific differences in far-field protein-surface interactions, neatly capturing the ability of ions to modulate the surface charge of muscovite that in turn selectively attracts one binding face of each protein over all others.

The co-self-assembly of proteins and nucleic acids (NAs) produces complex biomolecular machines (e.g., ribosomes and telomerases) that represent some of the most daunting targets for biomolecular design. Despite significant advances in protein and DNA or RNA nanotechnology, the construction of artificial nucleoprotein complexes has largely been limited to cases that rely on the NA-mediated spatial organization of protein units, rather than a cooperative interplay between protein- and NA-mediated interactions that typify natural nucleoprotein assemblies. We report here a structurally well-defined synthetic nucleoprotein assembly that forms through the synergy of three types of intermolecular interactions: Watson-Crick base pairing, NA-protein interactions, and protein-metal coordination. The fine thermodynamic balance between these interactions enables the formation of a crystalline architecture under highly specific conditions.

The sensitivity and responsiveness of living cells to environmental changes are enabled by dynamic protein structures, inspiring efforts to construct artificial supramolecular protein assemblies. However, despite their sophisticated structures, designed protein assemblies have yet to be incorporated into macroscale devices for real-life applications. We report a 2D crystalline protein assembly of ^C98/E57/E66L-rhamnulose-1-phosphate aldolase (^CEERhuA) that selectively blocks or passes molecular species when exposed to a chemical trigger. ^CEERhuA crystals are engineered via cobalt(II) coordination bonds to undergo a coherent conformational change from a closed state (pore dimensions <1 nm) to an ajar state (pore dimensions ~4 nm) when exposed to an HCN(g) trigger. When layered onto a mesoporous silicon (pSi) photonic crystal optical sensor configured to detect HCN_(g), the 2D ^CEERhuA crystal layer effectively blocks interferents that would otherwise result in a false positive signal. The 2D ^CEERhuA crystal layer opens in selective response to low-ppm levels of HCN_(g), allowing analyte penetration into the pSi sensor layer for detection. These findings illustrate that designed protein assemblies can function as dynamic components of solid-state devices in non-aqueous environments.