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 B 6 ). Here, we show that ConK catalyzes the desulfurization of cysteine to H 2 S, 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.
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A Strategy for Combinatorial Cavity Design in De Novo Proteins
Protein sequence space is vast; nature uses only an infinitesimal fraction of possible sequences to sustain life. Are there solutions to biological problems other than those provided by nature? Can we create artificial proteins that sustain life? To investigate these questions, we have created combinatorial collections, or libraries, of novel sequences with no homology to those found in living organisms. Previously designed libraries contained numerous functional proteins. However, they often formed dynamic, rather than well-ordered structures, which complicated structural and mechanistic characterization. To address this challenge, we describe the development of new libraries based on the de novo protein S-824, a 4-helix bundle with a very stable 3-dimensional structure. Distinct from previous libraries, we targeted variability to a specific region of the protein, seeking to create potential functional sites. By characterizing variant proteins from this library, we demonstrate that the S-824 scaffold tolerates diverse amino acid substitutions in a putative cavity, including buried polar residues suitable for catalysis. We designed and created a DNA library encoding 1.7 × 106 unique protein sequences. This new library of stable de novo α-helical proteins is well suited for screens and selections for a range of functional activities in vitro and in vivo.
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- Award ID(s):
- 1947720
- NSF-PAR ID:
- 10205656
- Date Published:
- Journal Name:
- Life
- Volume:
- 10
- Issue:
- 2
- ISSN:
- 2075-1729
- Page Range / eLocation ID:
- 9
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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William DeGrado (Ed.)more » « less
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. -
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The successful de novo design of proteins can provide insights into the physical chemical basis of stability, the role of evolution in constraining amino acid sequences, and the production of customizable platforms for engineering applications. Previous guanidine hydrochloride (GdnHCl; an ionic denaturant) experiments of a designed, naturally occurring βα fold, Di-III_14, revealed a cooperative, two-state unfolding transition and a modest stability. Continuous-flow mixing experiments in our laboratory revealed a simple two-state reaction in the microsecond to millisecond time range and consistent with the thermodynamic results. In striking contrast, the protein remains folded up to 9.25 M in urea, a neutral denaturant, and hydrogen exchange (HDX) NMR analysis in water revealed the presence of numerous high-energy states that interconvert on a time scale greater than seconds. The complex protection pattern for HDX corresponds closely with a pair of electrostatic networks on the surface and an extensive network of hydrophobic side chains in the interior of the protein. Mutational analysis showed that electrostatic and hydrophobic networks contribute to the resistance to urea denaturation for the WT protein; remarkably, single charge reversals on the protein surface restore the expected urea sensitivity. The roughness of the energy surface reflects the densely packed hydrophobic core; the removal of only two methyl groups eliminates the high-energy states and creates a smooth surface. The design of a very stable βα fold containing electrostatic and hydrophobic networks has created a complex energy surface rarely observed in natural proteins.
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Abstract While native proteins cover diverse structural spaces and achieve various biological events, not many of them can directly serve human needs. One reason is that the native proteins usually contain idiosyncrasies evolved for their native functions but disfavoring engineering requirements. To overcome this issue, one strategy is to create de novo proteins which are designed to possess improved stability, high environmental tolerance, and enhanced engineering potential. Compared to other protein engineering strategies, in silico design of de novo proteins has significantly expanded the protein structural and sequence spaces, reduced wet lab workload, and incorporated engineered features in a guided and efficient manner. In the Baker laboratory we have been applying a design pipeline that uses the blueprint builder to design different folds of de novo proteins, and have successfully obtained libraries of de novo proteins with improved stability and engineering potential. In this article, we will use the design of de novo β‐barrel proteins as an example to describe the principles and basic procedures of the blueprint builder−based design pipeline. © 2020 Wiley Periodicals LLC.
Basic Protocol 1 : The construction of blueprintsAlternate Protocol : Build blueprints based on existing protein.pdb filesBasic Protocol 2 : De novo protein design pipeline using the blueprint builder -
Abstract Background The explosive radiation and diversification of the advanced snakes (superfamily Colubroidea) was associated with changes in all aspects of the shared venom system. Morphological changes included the partitioning of the mixed ancestral glands into two discrete glands devoted for production of venom or mucous respectively, as well as changes in the location, size and structural elements of the venom-delivering teeth. Evidence also exists for homology among venom gland toxins expressed across the advanced snakes. However, despite the evolutionary novelty of snake venoms, in-depth toxin molecular evolutionary history reconstructions have been mostly limited to those types present in only two front-fanged snake families, Elapidae and Viperidae. To have a broader understanding of toxins shared among extant snakes, here we first sequenced the transcriptomes of eight taxonomically diverse rear-fanged species and four key viperid species and analysed major toxin types shared across the advanced snakes. Results Transcriptomes were constructed for the following families and species: Colubridae - Helicops leopardinus , Heterodon nasicus , Rhabdophis subminiatus ; Homalopsidae – Homalopsis buccata ; Lamprophiidae - Malpolon monspessulanus , Psammophis schokari , Psammophis subtaeniatus , Rhamphiophis oxyrhynchus ; and Viperidae – Bitis atropos , Pseudocerastes urarachnoides , Tropidolaeumus subannulatus , Vipera transcaucasiana . These sequences were combined with those from available databases of other species in order to facilitate a robust reconstruction of the molecular evolutionary history of the key toxin classes present in the venom of the last common ancestor of the advanced snakes, and thus present across the full diversity of colubroid snake venoms. In addition to differential rates of evolution in toxin classes between the snake lineages, these analyses revealed multiple instances of previously unknown instances of structural and functional convergences. Structural convergences included: the evolution of new cysteines to form heteromeric complexes, such as within kunitz peptides (the beta-bungarotoxin trait evolving on at least two occasions) and within SVMP enzymes (the P-IIId trait evolving on at least three occasions); and the C-terminal tail evolving on two separate occasions within the C-type natriuretic peptides, to create structural and functional analogues of the ANP/BNP tailed condition. Also shown was that the de novo evolution of new post-translationally liberated toxin families within the natriuretic peptide gene propeptide region occurred on at least five occasions, with novel functions ranging from induction of hypotension to post-synaptic neurotoxicity. Functional convergences included the following: multiple occasions of SVMP neofunctionalised in procoagulant venoms into activators of the clotting factors prothrombin and Factor X; multiple instances in procoagulant venoms where kunitz peptides were neofunctionalised into inhibitors of the clot destroying enzyme plasmin, thereby prolonging the half-life of the clots formed by the clotting activating enzymatic toxins; and multiple occasions of kunitz peptides neofunctionalised into neurotoxins acting on presynaptic targets, including twice just within Bungarus venoms. Conclusions We found novel convergences in both structural and functional evolution of snake toxins. These results provide a detailed roadmap for future work to elucidate predator–prey evolutionary arms races, ascertain differential clinical pathologies, as well as documenting rich biodiscovery resources for lead compounds in the drug design and discovery pipeline.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/life10020009
