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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 upregulates 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.

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 m 2 (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 central challenge of synthetic biology is to enable the growth of living systems using parts that are not derived from nature, but designed and synthesized in the laboratory. As an initial step toward achieving this goal, we probed the ability of a collection of >10(6) de novo designed proteins to provide biological functions necessary to sustain cell growth. Our collection of proteins was drawn from a combinatorial library of 102-residue sequences, designed by binary patterning of polar and nonpolar residues to fold into stable 4-helix bundles. We probed the capacity of proteins from this library to function in vivo by testing their abilities to rescue 27 different knockout strains of Escherichia coli, each deleted for a conditionally essential gene. Four different strains--ΔserB, ΔgltA, ΔilvA, and Δfes--were rescued by specific sequences from our library. Further experiments demonstrated that a strain simultaneously deleted for all four genes was rescued by co-expression of four novel sequences. Thus, cells deleted for ∼0.1% of the E. coli genome (and ∼1% of the genes required for growth under nutrient-poor conditions) can be sustained by sequences designed de novo.

The thermal and electrical conductivity probe (TECP), a component of the Microscopy, Electrochemistry, and Conductivity Analyzer (MECA), was included on the Phoenix Lander to conduct in situ measurements of the exchange of heat and water in the Martian polar terrain. TECP measured regolith thermal conductivity, heat capacity, temperature, electrical conductivity, and dielectric permittivity throughout the mission. A relative humidity sensor returned the first in situ humidity measurements from the Martian surface. The dry overburden above the ground ice is a good thermal insulator (average κ = 0.085 W m−1 K−1 and average Cρ = 1.05 × 106 J m−3 K−1). Surface thermal inertia (I) calculated from these values agrees well with daytime orbital determinations, but differences in the spatial and temporal scale of heat transport lead to very different measurements at night. Electrical conductivity was consistent with open circuit throughout the mission; an upper limit conductivity of 2 nS cm−1 is derived. Bulk dielectric permittivity (ɛb) shows several puzzling signals but also a systematic increase overnight in the latter half of the mission, contemporaneous with H2O adsorption. The magnitude of the increase is difficult to reconcile with expected changes in unfrozen water. Atmospheric H2O averages around 1.8 Pa during the day, corresponding to a RH < 5%. At night, much of the H2O disappears from the atmosphere, and RH increases to ∼100%. Temperature and H2O partial pressure data suggest that adsorption on mineral surfaces plays a major role in scrubbing H2O, with a possible contribution from perchlorate salts.

A primary objective of the Phoenix mission was to examine the characteristics of high latitude ground ice on Mars. We report observations of ground ice, its depth distribution and stability characteristics, and examine its origins and history. High latitude ground ice was explored through a dozen trench complexes and landing thruster pits, over a range of polygon morphological provinces. Shallow ground ice was found to be abundant under a layer of relatively loose ice‐free soil with a mean depth of 4.6 cm, which varied by more than 10x from trench to trench. These variations can be attributed mainly to slope effects and thermal inertia variations in the overburden soil affecting ground temperatures. The presence of ice at this depth is consistent with vapor‐diffusive equilibrium with respect to a mean atmospheric water content of 3.4 × 1019 m−3, consistent with the present‐day climate. Significant ice heterogeneity was observed, with two major forms: ice‐cemented soil and relatively pure light toned ice. Ice‐cemented soils, which comprised about 90% of the icy material exposed by trenching, are best explained as vapor deposited pore ice in a matrix supported porous soil. Light toned ice deposits represent a minority of the subsurface and are thought to consist of relatively thin near surface deposits. The origin of these relatively pure ice deposits appears most consistent with the formation of excess ice by soil ice segregation, such as would occur by thin film migration and the formation of ice lenses, needle ice, or similar ice structures.

Here, we present the first instance of utilizing de novo proteins to regulate the size of cadmium sulfide (CdS) quantum dots. Four proteins were found to bind to CdS and cap the growth of CdS quantum dots, leading to precise size control, as evidenced by absorbance and fluorescence spectra. Increasing the concentration of CdS does not change the absorbance and emission peaks, thereby indicating that the proteins effectively constrain the size of the quantum dots. Employing different proteins also yielded quantum dots with distinct optical and physical properties, including the appearance of biomediated nanorods when SynI3 was utilized. Moreover, the de novo proteins effectively maintained the stability of the quantum dots for up to 7 days, surpassing the stability of quantum dots capped by the small molecule, l-cysteine. The ability to cap CdS likely stems from their affinities for Cd2+, yet there does not seem to be a direct correlation between the affinity for Cd2+ and the size of resulting quantum dots.

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.

Amyloid fibrils are associated with a variety of neurodegenerative maladies including Alzheimer's disease and the prion diseases. The structures of amyloid fibrils are composed of β-strands oriented orthogonal to the fibril axis (\"cross β\" structure). We previously reported the design and characterization of a combinatorial library of de novo β-sheet proteins that self-assemble into fibrillar structures resembling amyloid. The libraries were designed by using a \"binary code\" strategy, in which the locations of polar and nonpolar residues are specified explicitly, but the identities of these residues are not specified and are varied combinatorially. The initial libraries were designed to encode proteins containing amphiphilic β-strands separated by reverse turns. Each β-strand was designed to be seven residues long, with polar (○) and nonpolar (●) amino acids arranged with an alternating periodicity (○●○●○●○). The initial design specified the identical polar/nonpolar pattern for all of the β-strands; no strand was explicitly designated to form the edges of the resulting β-sheets. With all β-strands preferring to occupy interior (as opposed to edge) locations, intermolecular oligomerization was favored, and the proteins assembled into amyloid-like fibrils. To assess whether explicit design of edge-favoring strands might tip the balance in favor of monomeric β-sheet proteins, we have now redesigned the first and/or last β-strands of several sequences from the original library. In the redesigned β-strands, the binary pattern is changed from ○●○●○●○ to ○●○K○●○ (K denotes lysine). The presence of a lysine on the nonpolar face of a β-strand should disfavor fibrillar structures because such structures would bury an uncompensated charge. The nonpolar → lysine mutations, therefore, would be expected to favor monomeric structures in which the ○●○K○●○ sequences form edge strands with the charged lysine side chain accessible to solvent. To test this hypothesis, we constructed several second generation sequences in which the central nonpolar residue of either the N-terminal β-strand or the C-terminal β-strand (or both) is changed to lysine. Characterization of the redesigned proteins shows that they form monomeric β-sheet 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 ~10 9 and a catalytic proficiency of >10 14  M −1 , comparable to larger enzymes shaped by billions of years of evolution. Evolution separates complex modern enzymes from their hypothetical simpler early ancestors, which raises the question of how unevolved sequences can develop new functions. Here a library of non-natural protein sequences was subjected to ultrahigh-throughput screens in microfluidic droplets, leading to the isolation of a phosphodiesterase enzyme capable of hydrolysing the biological second messenger, cyclic AMP.

One hundred years ago, Alois Alzheimer observed a relationship between cognitive impairment and the presence of plaque in the brains of patients suffering from the disease that bears his name. The plaque was subsequently shown to be composed primarily of a 42-residue peptide called amyloid β (Aβ) 42. Despite the importance of Aβ42 aggregation in the molecular etiology of Alzheimer's disease, the amino acid sequence determinants of this process have yet to be elucidated. Although stretches of hydrophobic residues in the C-terminal half of Aβ42 have been implicated, the mechanism by which these residues promote aggregation remains unclear. In particular, it is not known whether the side chains of these hydrophobic residues mediate specific interactions that direct self-assembly or, alternatively, whether hydrophobicity per se at these positions is sufficient to promote aggregation. To distinguish between these two possibilities, we substituted 12 hydrophobic residues in the C-terminal half of Aβ42 with random nonpolar residues. The mutant sequences were screened by using a fusion of Aβ42 to GFP. Because aggregation of Aβ42 prevents folding of the GFP reporter, this screen readily distinguishes aggregating from nonaggregating variants of Aβ42. Application of the screen demonstrated that, despite the presence of 8-12 mutations, all of the sequences aggregated. To confirm these results, several of the mutant sequences were prepared as synthetic peptides and shown to form amyloid fibrils similar to those of WT Aβ42. These findings indicate that hydrophobic stretches in the sequence of Aβ42, rather than specific side chains, are sufficient to promote aggregation.