Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions.

Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial ß sheet proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.

Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26-His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

A de novo protein catalyzes the synthesis of semiconductor quantum dots.

De novo proteins constructed from novel amino acid sequences are distinct from proteins that evolved in nature. Construct K (ConK) is a binary-patterned de novo designed protein that rescues Escherichia coli from otherwise toxic concentrations of copper. ConK was recently found to bind the cofactor PLP (pyridoxal phosphate, the active form of vitamin B6). Here, we show that ConK catalyzes the desulfurization of cysteine to H2S, which can be used to synthesize CdS nanocrystals in solution. The CdS nanocrystals are approximately 3 nm, as measured by transmission electron microscope, with optical properties similar to those seen in chemically synthesized quantum dots. The CdS nanocrystals synthesized using ConK have slower growth rates and a different growth mechanism than those synthesized using natural biomineralization pathways. The slower growth rate yields CdS nanocrystals with two desirable properties not observed during biomineralization using natural proteins. First, CdS nanocrystals are predominantly of the zinc blende crystal phase; this is in stark contrast to natural biomineralization routes that produce a mixture of zinc blende and wurtzite phase CdS. Second, in contrast to the growth and eventual precipitation observed in natural biomineralization systems, the CdS nanocrystals produced by ConK stabilize at a final size. Future optimization of CdS nanocrystal growth using ConK-or other de novo proteins-may help to overcome the limits on nanocrystal quality typically observed from natural biomineralization by enabling the synthesis of more stable, high-quality quantum dots at room temperature.

A Completely De Novo ATPase from Combinatorial Protein Design.

Our understanding of biological chemistry is shaped by the observation that all life comes from other life-as Pasteur put it, omne vivum ex vivo. A key step in expanding our biochemical vocabulary is to recapitulate biogenic catalysis using non-natural sequences that did not arise from common ancestry. Here we describe an enzyme designed completely de novo that hydrolyzes ATP. This protein was designed to lack ß-sheet structure and is competitively inhibited by magnesium, two traits that are unlike natural ATPases.

A de novo enzyme catalyzes a life-sustaining reaction in Escherichia coli.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Here we describe Syn-F4, the first de novo protein that meets both criteria. Purified Syn-F4 hydrolyzes the siderophore ferric enterobactin, and expression of Syn-F4 allows an inviable strain of Escherichia coli to grow in iron-limited medium. These findings demonstrate that entirely new sequences can provide life-sustaining enzymatic functions in living organisms.

A protein constructed de novo enables cell growth by altering gene regulation.

Recent advances in protein design rely on rational and computational approaches to create novel sequences that fold and function. In contrast, natural systems selected functional proteins without any design a priori. In an attempt to mimic nature, we used large libraries of novel sequences and selected for functional proteins that rescue Escherichia coli cells in which a conditionally essential gene has been deleted. In this way, the de novo protein SynSerB3 was selected as a rescuer of cells in which serB, which encodes phosphoserine phosphatase, an enzyme essential for serine biosynthesis, was deleted. However, SynSerB3 does not rescue the deleted activity by catalyzing hydrolysis of phosphoserine. Instead, SynSerB3 up-regulates hisB, a gene encoding histidinol phosphate phosphatase. This endogenous E. coli phosphatase has promiscuous activity that, when overexpressed, compensates for the deletion of phosphoserine phosphatase. Thus, the de novo protein SynSerB3 rescues the deletion of serB by altering the natural regulation of the His operon.

Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity.

When organisms are subjected to environmental challenges, including growth inhibitors and toxins, evolution often selects for the duplication of endogenous genes, whose overexpression can provide a selective advantage. Such events occur both in natural environments and in clinical settings. Microbial cells-with their large populations and short generation times-frequently evolve resistance to a range of antimicrobials. While microbial resistance to antibiotic drugs is well documented, less attention has been given to the genetic elements responsible for resistance to metal toxicity. To assess which overexpressed genes can endow gram-negative bacteria with resistance to metal toxicity, we transformed a collection of plasmids overexpressing all E. coli open reading frames (ORFs) into naive cells, and selected for survival in toxic concentrations of six transition metals: Cd, Co, Cu, Ni, Ag, Zn. These selections identified 48 hits. In each of these hits, the overexpression of an endogenous E. coli gene provided a selective advantage in the presence of at least one of the toxic metals. Surprisingly, the majority of these cases (28/48) were not previously known to function in metal resistance or homeostasis. These findings highlight the diverse mechanisms that biological systems can deploy to adapt to environments containing toxic concentrations of metals.

Self-Assembling Nano-Architectures Created from a Protein Nano-Building Block Using an Intermolecularly Folded Dimeric de Novo Protein.

The design of novel proteins that self-assemble into supramolecular complexes is an important step in the development of synthetic biology and nanotechnology. Recently, we described the three-dimensional structure of WA20, a de novo protein that forms an intermolecularly folded dimeric 4-helix bundle (PDB code 3VJF ). To harness the unusual intertwined structure of WA20 for the self-assembly of supramolecular nanostructures, we created a protein nanobuilding block (PN-Block), called WA20-foldon, by fusing the dimeric structure of WA20 to the trimeric foldon domain of fibritin from bacteriophage T4. The WA20-foldon fusion protein was expressed in the soluble fraction in Escherichia coli, purified, and shown to form several homooligomeric forms. The stable oligomeric forms were further purified and characterized by a range of biophysical techniques. Size exclusion chromatography, multiangle light scattering, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) analyses indicate that the small (S form), middle (M form), and large (L form) forms of the WA20-foldon oligomers exist as hexamer (6-mer), dodecamer (12-mer), and octadecamer (18-mer), respectively. These findings suggest that the oligomers in multiples of 6-mer are stably formed by fusing the interdigitated dimer of WA20 with the trimer of foldon domain. Pair-distance distribution functions obtained from the Fourier inversion of the SAXS data suggest that the S and M forms have barrel- and tetrahedron-like shapes, respectively. These results demonstrate that the de novo WA20-foldon is an effective building block for the creation of self-assembling artificial nanoarchitectures.

Selection of a promiscuous minimalist cAMP phosphodiesterase from a library of de novo designed proteins.

The ability of unevolved amino acid sequences to become biological catalysts was key to the emergence of life on Earth. However, billions of years of evolution separate complex modern enzymes from their simpler early ancestors. To probe how unevolved sequences can develop new functions, we use ultrahigh-throughput droplet microfluidics to screen for phosphoesterase activity amidst a library of more than one million sequences based on a de novo designed 4-helix bundle. Characterization of hits revealed that acquisition of function involved a large jump in sequence space enriching for truncations that removed >40% of the protein chain. Biophysical characterization of a catalytically active truncated protein revealed that it dimerizes into an α-helical structure, with the gain of function accompanied by increased structural dynamics. The identified phosphodiesterase is a manganese-dependent metalloenzyme that hydrolyses a range of phosphodiesters. It is most active towards cyclic AMP, with a rate acceleration of ~109 and a catalytic proficiency of >1014 M-1, comparable to larger enzymes shaped by billions of years of evolution.

Present-day thermal and water activity environment of the Mars Sample Return collection.

The Mars Sample Return mission intends to retrieve a sealed collection of rocks, regolith, and atmosphere sampled from Jezero Crater, Mars, by the NASA Perseverance rover mission. For all life-related research, it is necessary to evaluate water availability in the samples and on Mars. Within the first Martian year, Perseverance has acquired an estimated total mass of 355 g of rocks and regolith, and 38 µmoles of Martian atmospheric gas. Using in-situ observations acquired by the Perseverance rover, we show that the present-day environmental conditions at Jezero allow for the hydration of sulfates, chlorides, and perchlorates and the occasional formation of frost as well as a diurnal atmospheric-surface water exchange of 0.5-10 g water per m2 (assuming a well-mixed atmosphere). At night, when the temperature drops below 190 K, the surface water activity can exceed 0.5, the lowest limit for cell reproduction. During the day, when the temperature is above the cell replication limit of 245 K, water activity is less than 0.02. The environmental conditions at the surface of Jezero Crater, where these samples were acquired, are incompatible with the cell replication limits currently known on Earth.

A novel inhibitor of amyloid ß (Aß) peptide aggregation: from high throughput screening to efficacy in an animal model of Alzheimer disease.

Compelling evidence indicates that aggregation of the amyloid ß (Aß) peptide is a major underlying molecular culprit in Alzheimer disease. Specifically, soluble oligomers of the 42-residue peptide (Aß42) lead to a series of events that cause cellular dysfunction and neuronal death. Therefore, inhibiting Aß42 aggregation may be an effective strategy for the prevention and/or treatment of disease. We describe the implementation of a high throughput screen for inhibitors of Aß42 aggregation on a collection of 65,000 small molecules. Among several novel inhibitors isolated by the screen, compound D737 was most effective in inhibiting Aß42 aggregation and reducing Aß42-induced toxicity in cell culture. The protective activity of D737 was most significant in reducing the toxicity of high molecular weight oligomers of Aß42. The ability of D737 to prevent Aß42 aggregation protects against cellular dysfunction and reduces the production/accumulation of reactive oxygen species. Most importantly, treatment with D737 increases the life span and locomotive ability of flies in a Drosophila melanogaster model of Alzheimer disease.

Mars Oxygen ISRU Experiment (MOXIE)-Preparing for human Mars exploration.

MOXIE [Mars Oxygen In Situ Resource Utilization (ISRU) Experiment] is the first demonstration of ISRU on another planet, producing oxygen by solid oxide electrolysis of carbon dioxide in the martian atmosphere. A scaled-up MOXIE would contribute to sustainable human exploration of Mars by producing on-site the tens of tons of oxygen required for a rocket to transport astronauts off the surface of Mars, instead of having to launch hundreds of tons of material from Earth's surface to transport the required oxygen to Mars. MOXIE has produced oxygen seven times between landing in February 2021 and the end of 2021 and will continue to demonstrate oxygen production during night and day throughout all martian seasons. This paper reviews what MOXIE has accomplished and the implications for larger-scale oxygen-producing systems.

Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aß peptide.

The aggregation of polypeptides into amyloid fibrils is associated with a number of human diseases. Because these fibrils--or intermediates on the aggregation pathway--play important roles in the etiology of disease, considerable effort has been expended to understand which features of the amino acid sequence promote aggregation. One feature suspected to direct aggregation is the π-stacking of aromatic residues. Such π-stacking interactions have also been proposed as the targets for various aromatic compounds that are known to inhibit aggregation. In the case of Alzheimer's disease, the aromatic side chains Phe19 and Phe20 in the wild-type amyloid beta (Aß) peptide have been implicated. To explicitly test whether the aromaticity of these side chains plays a role in aggregation, we replaced these two phenylalanine side chains with leucines or isoleucines. These residues have similar sizes and hydrophobicities as Phe but are not capable of π-stacking. Thioflavin-T fluorescence and electron microscopy demonstrate that replacement of residues 19 and 20 by Leu or Ile did not prevent aggregation, but rather enhanced amyloid formation. Further experiments showed that aromatic inhibitors of aggregation are as effective against Ile- and Leu-substituted versions of Aß42 as they are against wild-type Aß. These results suggest that aromatic π-stacking interactions are not critical for Aß aggregation or for the inhibition of Aß aggregation.

Small molecule microarrays enable the discovery of compounds that bind the Alzheimer's Aß peptide and reduce its cytotoxicity.

The amyloid-ß (Aß) aggregation pathway is a key target in efforts to discover therapeutics that prevent or delay the onset of Alzheimer's disease. Efforts at rational drug design, however, are hampered by uncertainties about the precise nature of the toxic aggregate. In contrast, high-throughput screening of compound libraries does not require a detailed understanding of the structure of the toxic species, and can provide an unbiased method for the discovery of small molecules that may lead to effective therapeutics. Here, we show that small molecule microarrays (SMMs) represent a particularly promising tool for identifying compounds that bind the Aß peptide. Microarray slides with thousands of compounds immobilized on their surface were screened for binding to fluorescently labeled Aß. Seventy-nine compounds were identified by the SMM screen, and then assayed for their ability to inhibit the Aß-induced killing of PC12 cells. Further experiments focused on exploring the mechanism of rescue for one of these compounds: Electron microscopy and Congo red binding showed that the compound enhances fibril formation, and suggest that it may rescue cells by accelerating Aß aggregation past an early toxic oligomer. These findings demonstrate that the SMM screen for binding to Aß is effective at identifying compounds that reduce Aß toxicity, and can reveal potential therapeutic leads without the biases inherent in methods that focus on inhibitors of aggregation.

Hyperstable De Novo Protein with a Dimeric Bisecting Topology.

Recently, we designed and assembled protein nanobuilding blocks (PN-Blocks) from an intermolecularly folded dimeric de novo protein called WA20. Using this dimeric 4-helix bundle, we constructed a series of self-assembling supramolecular nanostructures including polyhedra and chain-type complexes. Here we describe the stabilization of WA20 by designing mutations that stabilize the helices and hydrophobic core. The redesigned proteins denature with substantially higher midpoints, with the most stable variant, called Super WA20 (SUWA), displaying an extremely high midpoint (Tm = 122 °C), much higher than the Tm of WA20 (75 °C). The crystal structure of SUWA reveals an intermolecularly folded dimer with bisecting U topology, similar to the parental WA20 structure, with two long α-helices of a protomer intertwined with the helices of another protomer. Molecular dynamics simulations demonstrate that the redesigned hydrophobic core in the center of SUWA significantly suppresses the deformation of helices observed in the same region of WA20, suggesting this is a critical factor stabilizing the SUWA structure. This hyperstable de novo protein is expected to be useful as nanoscale pillars of PN-Block components in new types of self-assembling nanoarchitectures.

Stability of Protein Structure during Nanocarrier Encapsulation: Insights on Solvent Effects from Simulations and Spectroscopic Analysis.

The dosing of peptide and protein therapeutics is complicated by rapid clearance from the blood pool and poor cellular membrane permeability. Encapsulation into nanocarriers such as liposomes or polymersomes has long been explored to overcome these limitations, but manufacturing challenges have limited clinical translation by these approaches. Recently, inverse Flash NanoPrecipitation (iFNP) has been developed to produce highly loaded polymeric nanocarriers with the peptide or protein contained within a hydrophilic core, stabilized by a hydrophobic polymer shell. Encapsulation of proteins with higher-order structure requires understanding how processing may affect their conformational state. We demonstrate a combined experimental/simulation approach to characterize protein behavior during iFNP processing steps using the Trp-cage protein TC5b as a model. Explicit-solvent fully atomistic molecular dynamics simulations with enhanced sampling techniques are coupled with two-dimensional heteronuclear multiple-quantum coherence nuclear magnetic resonance spectroscopy (2D-HMQC NMR) and circular dichroism to determine the structure of TC5b during mixed-solvent exposure encountered in iFNP processing. The simulations involve atomistic models of mixed solvents and protein to capture the complexity of the hydrogen bonding and hydrophobic interactions between water, dimethylsulfoxide (DMSO), and the protein. The combined analyses reveal structural unfolding of the protein in 11 M DMSO but confirm complete refolding after release from the polymeric nanocarrier back into an aqueous phase. These results highlight the insights that simulations and NMR provide for the formulation of proteins in nanocarriers.

Unevolved De Novo Proteins Have Innate Tendencies to Bind Transition Metals.

Life as we know it would not exist without the ability of protein sequences to bind metal ions. Transition metals, in particular, play essential roles in a wide range of structural and catalytic functions. The ubiquitous occurrence of metalloproteins in all organisms leads one to ask whether metal binding is an evolved trait that occurred only rarely in ancestral sequences, or alternatively, whether it is an innate property of amino acid sequences, occurring frequently in unevolved sequence space. To address this question, we studied 52 proteins from a combinatorial library of novel sequences designed to fold into 4-helix bundles. Although these sequences were neither designed nor evolved to bind metals, the majority of them have innate tendencies to bind the transition metals copper, cobalt, and zinc with high nanomolar to low-micromolar affinity.

Are natural proteins special? Can we do that?

Natural proteins represent a minuscule fraction of possible sequence space. These very rare sequences display remarkable properties: They fold into many different stable structures, and perform a wide range of complex biological functions. These two considerations-rarity and functionality-may suggest that natural proteins are somehow special. Is this true? We address this question by exploring attempts to recapitulate the special structures and functions of natural proteins into sequences designed de novo.

Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks.

The design of novel proteins that self-assemble into supramolecular complexes is important for development in nanobiotechnology and synthetic biology. Recently, we designed and created a protein nanobuilding block (PN-Block), WA20-foldon, by fusing an intermolecularly folded dimeric de novo WA20 protein and a trimeric foldon domain of T4 phage fibritin (Kobayashi et al., J. Am. Chem. Soc. 2015, 137, 11285). WA20-foldon formed several types of self-assembling nanoarchitectures in multiples of 6-mers, including a barrel-like hexamer and a tetrahedron-like dodecamer. In this study, to construct chain-like polymeric nanostructures, we designed de novo extender protein nanobuilding blocks (ePN-Blocks) by tandemly fusing two de novo binary-patterned WA20 proteins with various linkers. The ePN-Blocks with long helical linkers or flexible linkers were expressed in soluble fractions of Escherichia coli, and the purified ePN-Blocks were analyzed by native PAGE, size exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS), and transmission electron microscopy. These results suggest formation of various structural homo-oligomers. Subsequently, we reconstructed hetero-oligomeric complexes from extender and stopper PN-Blocks by denaturation and refolding. The present SEC-MALS and SAXS analyses show that extender and stopper PN-Block (esPN-Block) heterocomplexes formed different types of extended chain-like conformations depending on their linker types. Moreover, atomic force microscopy imaging in liquid suggests that the esPN-Block heterocomplexes with metal ions further self-assembled into supramolecular nanostructures on mica surfaces. Taken together, the present data demonstrate that the design and construction of self-assembling PN-Blocks using de novo proteins is a useful strategy for building polymeric nanoarchitectures of supramolecular protein complexes.

Protein design by binary patterning of polar and nonpolar amino acids.

The design of large libraries of well-folded de novo proteins is a powerful approach toward the ultimate goal of producing proteins with novel structures and functions for use in industry or medicine. A method for library design that incorporates both rational design and combinatorial diversity relies on the "binary patterning" of polar and nonpolar amino acids. Binary patterning is based on the premise that the appropriate arrangement of polar and nonpolar residues can direct a polypeptide chain to fold into amphipathic elements of secondary structure that anneal together to form a desired tertiary structure. A designed binary pattern exploits the periodicities inherent in protein secondary structure, and allows the identity of the side chain at each polar and nonpolar position to be varied combinatorially. This chapter provides an overview of the considerations necessary to use binary patterning to design libraries of novel proteins.