Constructing protein polyhedra via orthogonal chemical interactions.

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 design2-5. 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 architectures1,6-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 architectures7,8 and are, to our knowledge, unique among designed systems9-13 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.

Ion-dependent protein-surface interactions from intrinsic solvent response.

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.

Determining the Structural and Energetic Basis of Allostery in a De Novo Designed Metalloprotein Assembly.

Despite significant progress in protein design, the construction of protein assemblies that display complex functions (e.g., catalysis or allostery) remains a significant challenge. We recently reported the de novo construction of an allosteric supramolecular protein assembly (Zn-C38/C81/C96R14) in which the dissociation and binding of ZnII ions were coupled over a distance of 15 Å to the selective hydrolytic breakage and formation of a single disulfide bond. Zn-C38/C81/C96R14 was constructed by ZnII-templated assembly of a monomeric protein (R1, a derivative of cytochrome cb562) into a tetramer, followed by progressive incorporation of noncovalent and disulfide bonding interactions into the protein-protein interfaces to create a strained quaternary architecture. The interfacial strain thus built allowed mechanical coupling between the binding/dissociation of ZnII and formation/hydrolysis of a single disulfide bond (C38-C38) out of a possible six. While the earlier study provided structural evidence for the two end-states of allosteric coupling, the energetic basis for allosteric coupling and the minimal structural requirements for building this allosteric system were not understood. Toward this end, we have characterized the structures and Zn-binding properties of two related protein constructs (C38/C96R1 and C38R1) which also possess C38-C38 disulfide bonds. In addition, we have carried out extensive molecular dynamics simulations of C38/C81/C96R14 to understand the energetic basis for the selective cleavage of the C38-C38 disulfide bond upon ZnII dissociation. Our analyses reveal that the local interfacial environment around the C38-C38 bond is key to its selective cleavage, but this cleavage is only possible within the context of a stable quaternary architecture which enables structural coupling between ZnII coordination and the protein-protein interfaces.

Discrete Orientations of Interfacial Waters Direct Crystallization of Mica-Binding Proteins.

Understanding the basis of templated molecular assembly on a solid surface requires a fundamental comprehension of both short- and long-range aqueous response to the surface under a variety of solution conditions. Herein we provide a detailed picture of how the molecular-scale response to different mica surfaces yields distinct solvent orientations that produce quasi-static directional potentials onto which macromolecules can adsorb. We connect this directionality to observed (a)symmetric epitaxial alignment of designed proteins onto these surfaces, corroborate our findings with 3D atomic force microscopy experiments, and identify slight differences in surface structure as the origin of this effect. Our work provides a detailed picture of the intrinsic electrolyte response in the vicinity of mineral interfaces, with clear predictions for experiment, and highlights the role of solvent on the predictive assembly of hierarchical materials on mineral surfaces.

Design principles of protein switches.

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.

Assembly of a patchy protein into variable 2D lattices via tunable multiscale interactions.

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.

Self-Assembly of a Designed Nucleoprotein Architecture through Multimodal Interactions.

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.