Enhancing and shaping the immunogenicity of native-like HIV-1 envelope trimers with a two-component protein nanoparticle.

The development of native-like HIV-1 envelope (Env) trimer antigens has enabled the induction of neutralizing antibody (NAb) responses against neutralization-resistant HIV-1 strains in animal models. However, NAb responses are relatively weak and narrow in specificity. Displaying antigens in a multivalent fashion on nanoparticles (NPs) is an established strategy to increase their immunogenicity. Here we present the design and characterization of two-component protein NPs displaying 20 stabilized SOSIP trimers from various HIV-1 strains. The two-component nature permits the incorporation of exclusively well-folded, native-like Env trimers into NPs that self-assemble in vitro with high efficiency. Immunization studies show that the NPs are particularly efficacious as priming immunogens, improve the quality of the Ab response over a conventional one-component nanoparticle system, and are most effective when SOSIP trimers with an apex-proximate neutralizing epitope are displayed. Their ability to enhance and shape the immunogenicity of SOSIP trimers make these NPs a promising immunogen platform.

Multimerization of an Alcohol Dehydrogenase by Fusion to a Designed Self-Assembling Protein Results in Enhanced Bioelectrocatalytic Operational Stability.

Proteins designed for supramolecular assembly provide a simple means to immobilize and organize enzymes for biotechnology applications. We have genetically fused the thermostable alcohol dehydrogenase D (AdhD) from Pyrococcus furiosus to a computationally designed cage-forming protein (O3-33). The trimeric form of the O3-33-AdhD fusion protein was most active in solution. The immobilization of the fusion protein on bioelectrodes leads to a doubling of the electrochemical operational stability as compared to the unfused control proteins. Thus, the fusion of enzymes to the designed self-assembling domains offers a simple strategy to increase the stability in biocatalytic systems.

Evolution of a designed protein assembly encapsulating its own RNA genome.

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.

Accurate design of megadalton-scale two-component icosahedral protein complexes.

Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

Design of a hyperstable 60-subunit protein dodecahedron. [corrected].

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.

Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression.

We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.

Computational protein design enables a novel one-carbon assimilation pathway.

We describe a computationally designed enzyme, formolase (FLS), which catalyzes the carboligation of three one-carbon formaldehyde molecules into one three-carbon dihydroxyacetone molecule. The existence of FLS enables the design of a new carbon fixation pathway, the formolase pathway, consisting of a small number of thermodynamically favorable chemical transformations that convert formate into a three-carbon sugar in central metabolism. The formolase pathway is predicted to use carbon more efficiently and with less backward flux than any naturally occurring one-carbon assimilation pathway. When supplemented with enzymes carrying out the other steps in the pathway, FLS converts formate into dihydroxyacetone phosphate and other central metabolites in vitro. These results demonstrate how modern protein engineering and design tools can facilitate the construction of a completely new biosynthetic pathway.

The conserved PFT1 tandem repeat is crucial for proper flowering in Arabidopsis thaliana.

It is widely appreciated that short tandem repeat (STR) variation underlies substantial phenotypic variation in organisms. Some propose that the high mutation rates of STRs in functional genomic regions facilitate evolutionary adaptation. Despite their high mutation rate, some STRs show little to no variation in populations. One such STR occurs in the Arabidopsis thaliana gene PFT1 (MED25), where it encodes an interrupted polyglutamine tract. Although the PFT1 STR is large (∼270 bp), and thus expected to be extremely variable, it shows only minuscule variation across A. thaliana strains. We hypothesized that the PFT1 STR is under selective constraint, due to previously undescribed roles in PFT1 function. We investigated this hypothesis using plants expressing transgenic PFT1 constructs with either an endogenous STR or synthetic STRs of varying length. Transgenic plants carrying the endogenous PFT1 STR generally performed best in complementing a pft1 null mutant across adult PFT1-dependent traits. In stark contrast, transgenic plants carrying a PFT1 transgene lacking the STR phenocopied a pft1 loss-of-function mutant for flowering time phenotypes and were generally hypomorphic for other traits, establishing the functional importance of this domain. Transgenic plants carrying various synthetic constructs occupied the phenotypic space between wild-type and pft1 loss-of-function mutants. By varying PFT1 STR length, we discovered that PFT1 can act as either an activator or repressor of flowering in a photoperiod-dependent manner. We conclude that the PFT1 STR is constrained to its approximate wild-type length by its various functional requirements. Our study implies that there is strong selection on STRs not only to generate allelic diversity, but also to maintain certain lengths pursuant to optimal molecular function.

Accurate design of co-assembling multi-component protein nanomaterials.

The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.

Background-dependent effects of polyglutamine variation in the Arabidopsis thaliana gene ELF3.

Tandem repeats (TRs) have extremely high mutation rates and are often considered to be neutrally evolving DNA. However, in coding regions, TR copy number mutations can significantly affect phenotype and may facilitate rapid adaptation to new environments. In several human genes, TR copy number mutations that expand polyglutamine (polyQ) tracts beyond a certain threshold cause incurable neurodegenerative diseases. PolyQ-containing proteins exist at a considerable frequency in eukaryotes, yet the phenotypic consequences of natural variation in polyQ tracts that are not associated with disease remain largely unknown. Here, we use Arabidopsis thaliana to dissect the phenotypic consequences of natural variation in the polyQ tract encoded by EARLY FLOWERING 3 (ELF3), a key developmental gene. Changing ELF3 polyQ tract length affected complex ELF3-dependent phenotypes in a striking and nonlinear manner. Some natural ELF3 polyQ variants phenocopied elf3 loss-of-function mutants in a common reference background, although they are functional in their native genetic backgrounds. To test the existence of background-specific modifiers, we compared the phenotypic effects of ELF3 polyQ variants between two divergent backgrounds, Col and Ws, and found dramatic differences. In fact, the Col-ELF3 allele, encoding the shortest known ELF3 polyQ tract, was haploinsufficient in Ws × Col F(1) hybrids. Our data support a model in which variable polyQ tracts drive adaptation to internal genetic environments.

Increased Diels-Alderase activity through backbone remodeling guided by Foldit players.

Computational enzyme design holds promise for the production of renewable fuels, drugs and chemicals. De novo enzyme design has generated catalysts for several reactions, but with lower catalytic efficiencies than naturally occurring enzymes. Here we report the use of game-driven crowdsourcing to enhance the activity of a computationally designed enzyme through the functional remodeling of its structure. Players of the online game Foldit were challenged to remodel the backbone of a computationally designed bimolecular Diels-Alderase to enable additional interactions with substrates. Several iterations of design and characterization generated a 24-residue helix-turn-helix motif, including a 13-residue insertion, that increased enzyme activity >18-fold. X-ray crystallography showed that the large insertion adopts a helix-turn-helix structure positioned as in the Foldit model. These results demonstrate that human creativity can extend beyond the macroscopic challenges encountered in everyday life to molecular-scale design problems.