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The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

Natural nanomachines like the F1/F0-ATPase contain protein components that undergo rotation relative to each other. Designing such mechanically constrained nanoscale protein architectures with internal degrees of freedom is an outstanding challenge for computational protein design. Here we explore the de novo construction of protein rotary machinery from designed axle and ring components. Using cryoelectron microscopy, we find that axle-ring systems assemble as designed and populate diverse rotational states depending on symmetry match or mismatch and the designed interface energy landscape. These mechanical systems with internal rotational degrees of freedom are a step towards the systematic design of genetically encodable nanomachines.One-Sentence SummaryComputationally designed self-assembling protein rotary machines sample internal degrees of freedom sculpted within the energy landscape.

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects1–4. To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examinedin vivo, superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili., Peptide-based filamentous assemblies are successfully used for generation of structurally ordered materials, but their de novo design and structural characterization is challenging. Here, the authors provide a strategy for the design of self-assembling peptide nanotubes based on modifications of an arginine clasp interaction motif, and report the cryo-EM structures of seven designed nanotubes.

Design of a structurally defined helical assembly is described that involves recoding of the amino acid sequence of peptide GCN4-pAA. In solution and the crystalline state, GCN4-pAA adopts a 7-helix bundle structure that resembles a supramolecular lock washer. Structurally informed mutagenesis of the sequence of GCN4-pAA afforded peptide 7HSAP1, which undergoes self-association into a nanotube via noncovalent interactions between complementary interfaces of the coiled-coil lock-washer structures. Biophysical measurements conducted in solution and the solid state over multiple length scales of structural hierarchy are consistent with self-assembly of nanotube structures derived from 7-helix bundle subunits. The dimensions of the supramolecular assemblies are similar to those observed in the crystal structure of GCN4-pAA. Fluorescence studies of the interaction of 7HSAP1 with the solvatochromic fluorophore PRODAN indicated that the nanotubes could encapsulate shape-appropriate small molecules with high binding affinity.

The dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

Building with designed proteins General design principles for protein interaction specificity are challenging to extract. DNA nanotechnology, on the other hand, has harnessed the limited set of hydrogen-bonding interactions from Watson-Crick base-pairing to design and build a wide range of shapes. Protein-based materials have the potential for even greater geometric and chemical diversity, including additional functionality. Boyken et al. designed a class of protein oligomers that have interaction specificity determined by modular arrays of extensive hydrogen bond networks (see the Perspective by Netzer and Fleishman). They use the approach, which could one day become programmable, to build novel topologies with two concentric rings of helices. Science , this issue p. 680 ; see also p. 657

Building with alphahelical coiled coils Understanding how proteins fold into well-defined three-dimensional structures has been a longstanding challenge. Increased understanding has led to increased success at designing proteins that mimic existing protein folds. This raises the possibility of custom design of proteins with structures not seen in nature. Thomson et al. describe the design of channelcontaining α-helical barrels, and Huang et al. designed hyperstable helical bundles. Both groups used rational and computational design to make new protein structures based on α-helical coiled coils but took different routes to reach different target structures. Science , this issue p. 485 , p. 481

A collagen-mimetic peptide, NSIII, has been designed with three sequential blocks having positive, neutral, and negative charges, respectively. The non-canonical imino acid, (2S,4S)-4-aminoproline (amp), was used to specify the positive charges at the Xaa positions of (Xaa-Yaa-Gly) triads in the N-terminal domain of NSIII. Peptide NSIII underwent self-assembly from aqueous solution to form a highly homogeneous population of nanosheets. Two-dimensional crystalline sheets formed in which the length of the peptide defined the height of the sheets. These results contrasted with prior results on a similar multi-domain collagen-mimetic polypeptides in which the sheets had highly polydisperse distribution of sizes in the (x/y)- and (z)-dimensions. The structural differences between the two nanosheet assemblies were interpreted in terms of the relative stereoelectronic effects of the different aminoproline derivatives on the local triple helical conformation of the peptides.

We report the design of two collagen-mimetic peptide sequences, NSI and NSII, that self-assemble into structurally defined nanoscale sheets. The underlying structure of these nanosheets can be understood in terms of the layered packing of collagen triple helices in two dimensions. These nanosheet assemblies represent a novel morphology for collagen-based materials, which, on the basis of their defined structure, may be envisioned as potentially biocompatible platforms for controlled presentation of chemical functionality at the nanoscale. The molecularly programmed self-assembly of peptides NSI and NSII into nanosheets suggests that sequence-specific macromolecules offer significant promise as design elements for two-dimensional (2D) assemblies. This investigation provides a design rubric for fabrication of structurally defined, peptide-based nanosheets using the principles of solution-based self-assembly facilitated through complementary electrostatic interactions.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.