A large fraction of proteins naturally exist as symmetrical homooligomers or homopolymers 1. 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 different symmetry elements needed to form higher-order architectures 1,6-a daunting task for protein design. Here we introduce an inorganic chemical approach to address this outstanding problem, 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 Zn-binding motifs assembles through concurrent Fe 3+ and Zn 2+ coordination into discrete dodecameric and hexameric cages. Closely resembling natural polyhedral protein architectures 7,8 and unique among designed systems 9-13 , our artificial cages possess tightly packed shells devoid of large apertures, yet they can assemble and disassemble in response to diverse stimuli owing to their heterobimetallic construction on minimal interproteinbonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomer] to [8 Fe:21 Zn:12 protomer], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.NMDA receptors | ctenophores | crystal structures | evolution I n the nervous system and neuromuscular junction of many animal species, the amino acid L-glutamate acts as an excitatory neurotransmitter. The molecular organization of glutamate receptor ion channel (iGluR) subunits into an amino terminal domain (ATD), and a ligand binding domain (LBD) bisected by insertion of a pore loop ion channel generates a unique structural signature, distinct from that for other neurotransmitter receptors, that is easily identified by sequence analysis. Using this approach, hundreds of iGluR homologs are emerging from genome sequencing projects (1-5). Virtually all of these are glutamate receptors in name only; their functional properties, physiological function, and the ligands they bind have yet to be determined. Recent large-scale sequencing projects, which place ctenophores as candidates for the earliest metazoan lineage, reveal that iGluR homologs are abundantly represented in the genomes of the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, suggesting that glutamate was selected to act as a neurotransmitter very early in evolution (4, 5). The muscle cells of P. bachei respond to application of glutamate with action potential generation and both species have neural networks and exhibit complex predatory behaviors that might also be generated by iGluR activity (4, 5). However, as for most species studied in sequencing projects, ctenophore iGluRs have yet to be characterized.By contrast to our primitive state of knowledge for iGluRs recently discovered by genome sequencing projects, the iGluRs of vertebrate species have been extensively characterized, and based on their ligand binding properties, amino acid sequences...

De novo design and construction of stimuli-responsive protein assemblies that predictably switch between discrete conformational states remains an essential but highly challenging goal in biomolecular design. We previously reported synthetic, two-dimensional protein lattices self-assembled via disulfide bonding interactions, which endows them with a unique capacity to undergo coherent conformational changes without losing crystalline order. Here, we carried out all-atom molecular dynamics simulations to map the free-energy landscape of these lattices, validated this landscape through extensive structural characterization by electron microscopy and established that it is predominantly governed by solvent reorganization entropy. Subsequent redesign of the protein surface with conditionally repulsive electrostatic interactions enabled us to predictably perturb the free-energy landscape and obtain a new protein lattice whose conformational dynamics can be chemically and mechanically toggled between three different states with varying porosities and molecular densities.

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.

The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi. The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligandbinding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.glutamate receptors | X-ray crystallography | electrophysiology | molecular dynamics simulations | free energy calculations