De novo design of protein logic gates Chen, Zibo; Kibler, Ryan D; Hunt, Andrew ...
Science (American Association for the Advancement of Science),
04/2020, Letnik:
368, Številka:
6486
Journal Article
Recenzirano
Odprti dostop
The design of modular protein logic for regulating protein function at the posttranscriptional level is a challenge for synthetic biology. Here, we describe the design of two-input AND, OR, NAND, ...NOR, XNOR, and NOT gates built from de novo-designed proteins. These gates regulate the association of arbitrary protein units ranging from split enzymes to transcriptional machinery in vitro, in yeast and in primary human T cells, where they control the expression of the
gene related to T cell exhaustion. Designed binding interaction cooperativity, confirmed by native mass spectrometry, makes the gates largely insensitive to stoichiometric imbalances in the inputs, and the modularity of the approach enables ready extension to three-input OR, AND, and disjunctive normal form gates. The modularity and cooperativity of the control elements, coupled with the ability to de novo design an essentially unlimited number of protein components, should enable the design of sophisticated posttranslational control logic over a wide range of biological functions.
Single-cell RNA sequencing (scRNA-seq) has become an essential tool for characterizing gene expression in eukaryotes, but current methods are incompatible with bacteria. Here, we introduce microSPLiT ...(microbial split-pool ligation transcriptomics), a high-throughput scRNA-seq method for Gram-negative and Gram-positive bacteria that can resolve heterogeneous transcriptional states. We applied microSPLiT to >25,000
cells sampled at different growth stages, creating an atlas of changes in metabolism and lifestyle. We retrieved detailed gene expression profiles associated with known, but rare, states such as competence and prophage induction and also identified unexpected gene expression states, including the heterogeneous activation of a niche metabolic pathway in a subpopulation of cells. MicroSPLiT paves the way to high-throughput analysis of gene expression in bacterial communities that are otherwise not amenable to single-cell analysis, such as natural microbiota.
De novo protein design methods can create proteins with folds not yet seen in nature. These methods largely focus on optimizing the compatibility between the designed sequence and the intended ...conformation, without explicit consideration of protein folding pathways. Deeply knotted proteins, whose topologies may introduce substantial barriers to folding, thus represent an interesting test case for protein design. Here we report our attempts to design proteins with trefoil (3
) and pentafoil (5
) knotted topologies. We extended previously described algorithms for tandem repeat protein design in order to construct deeply knotted backbones and matching designed repeat sequences (N = 3 repeats for the trefoil and N = 5 for the pentafoil). We confirmed the intended conformation for the trefoil design by X ray crystallography, and we report here on this protein's structure, stability, and folding behaviour. The pentafoil design misfolded into an asymmetric structure (despite a 5-fold symmetric sequence); two of the four repeat-repeat units matched the designed backbone while the other two diverged to form local contacts, leading to a trefoil rather than pentafoil knotted topology. Our results also provide insights into the folding of knotted proteins.
Summary
Ruminococcus bromii is a dominant member of the human colonic microbiota that plays a ‘keystone’ role in degrading dietary resistant starch. Recent evidence from one strain has uncovered a ...unique cell surface ‘amylosome’ complex that organizes starch‐degrading enzymes. New genome analysis presented here reveals further features of this complex and shows remarkable conservation of amylosome components between human colonic strains from three different continents and a R. bromii strain from the rumen of Australian cattle. These R. bromii strains encode a narrow spectrum of carbohydrate active enzymes (CAZymes) that reflect extreme specialization in starch utilization. Starch hydrolysis products are taken up mainly as oligosaccharides, with only one strain able to grow on glucose. The human strains, but not the rumen strain, also possess transporters that allow growth on galactose and fructose. R. bromii strains possess a full complement of sporulation and spore germination genes and we demonstrate the ability to form spores that survive exposure to air. Spore formation is likely to be a critical factor in the ecology of this nutritionally highly specialized bacterium, which was previously regarded as ‘non‐sporing’, helping to explain its widespread occurrence in the gut microbiota through the ability to transmit between hosts.
A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The ...design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures
. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.
While direct cell transplantation holds great promise in treating many debilitating diseases, poor cell survival and engraftment following injection have limited effective clinical translation. ...Though injectable biomaterials offer protection against membrane‐damaging extensional flow and supply a supportive 3D environment in vivo that ultimately improves cell retention and therapeutic costs, most are created from synthetic or naturally harvested polymers that are immunogenic and/or chemically ill‐defined. This work presents a shear‐thinning and self‐healing telechelic recombinant protein‐based hydrogel designed around XTEN – a well‐expressible, non‐immunogenic, and intrinsically disordered polypeptide previously evolved as a genetically encoded alternative to PEGylation to “eXTENd” the in vivo half‐life of fused protein therapeutics. By flanking XTEN with self‐associating coil domains derived from cartilage oligomeric matrix protein, single‐component physically crosslinked hydrogels exhibiting rapid shear thinning and self‐healing through homopentameric coiled‐coil bundling are formed. Individual and combined point mutations that variably stabilize coil association enables a straightforward method to genetically program material viscoelasticity and biodegradability. Finally, these materials protect and sustain viability of encapsulated human fibroblasts, hepatocytes, embryonic kidney (HEK), and embryonic stem‐cell‐derived cardiomyocytes (hESC‐CMs) through culture, injection, and transcutaneous implantation in mice. These injectable XTEN‐based hydrogels show promise for both in vitro cell culture and in vivo cell transplantation applications.
Shear‐thinning and self‐healing telechelic recombinant protein‐based hydrogels based on XTEN are introduced. Point mutations to self‐associating coil domains yield materials with programmable viscoelasticity and biodegradability. These materials protect and sustain viability of human fibroblasts, hepatocytes, HEK, and embryonic stem‐cell‐derived cardiomyocytes cultured within these genetically encodable biopolymer networks following in vivo injection. Such injectable hydrogels hold promise for therapeutic cell transplantation.
Current numerical methods for simulating biophysical processes in aquatic environments are typically constructed in a grid-based Eulerian framework or as an individual-based model in a particle-based ...Lagrangian framework. Often, the biogeochemical processes and physical (hydrodynamic) processes occur at different time and space scales, and changes in biological processes do not affect the hydrodynamic conditions. Therefore, it is possible to develop an alternative strategy to grid-based approaches for linking hydrodynamic and biogeochemical models that can significantly improve computational efficiency for this type of linked biophysical model. In this work, we utilize a new technique that links hydrodynamic effects and biological processes through a property-carrying particle model (PCPM) in a Lagrangian/Eulerian framework. The model is tested in idealized cases and its utility is demonstrated in a practical application to Sandusky Bay. Results show the integration of Lagrangian and Eulerian approaches allows for a natural coupling of mass transport (represented by particle movements and random walk) and biological processes in water columns which is described by a nutrient-phytoplankton-zooplankton-detritus (NPZD) biological model. This method is far more efficient than traditional tracer-based Eulerian biophysical models for 3-D simulation, particularly for a large domain and/or ensemble simulations.
The abiotic fate of dissolved free amino acids considerably contributes to the cycling of dissolved sulfur and nitrogen in natural aquatic environments. However, the roles of the functional groups of ...chromophoric dissolved organic matter (CDOM) and the fate of free amino acids under sunlight irradiation in fresh waters are not fully understood. This study aims to elucidate the fate of photolabile methionine in the presence of three CDOM surrogate compounds, i.e., 1,4-naphthoquinone, 2-naphthaldehyde, and umbelliferone, and two standard CDOM by coupling experimental measurement, quantum chemical computations, and kinetic modeling. Results indicate that excited triplet-state CDOM and hydroxyl radicals are able to cleave the C–S bond in methionine, resulting in the formation of smaller amino acids and volatile sulfur-containing compounds. Singlet oxygen forms methionine sulfoxide and methionine sulfone. The distribution of phototransformation products offers an improved understanding of the fate of nitrogen- and sulfur-containing compounds and their uptake by microorganisms in natural aquatic environments.
Reverse osmosis (RO) is a membrane technology that separates dissolved species from water. RO has been applied for the removal of chemical contaminants from water for potable reuse applications. The ...presence of a wide variety of influent chemical contaminants and the insufficient rejection of low-molecular-weight neutral organics by RO calls for the need to develop a model that predicts the rejection of various organics. In this study, we develop a group contribution method (GCM) to predict the mass transfer coefficients by fragmenting the structure of low-molecular-weight neutral organics into small parts that interact with the RO membrane. Overall, 54 organics including 26 halogenated and oxygenated alkanes, 8 alkenes, and 20 alkyl and halobenzenes were used to determine 39 parameters to calibrate for 6 different RO membranes, including 4 brackish water and 2 seawater membranes. Through six membranes, approximately 80% of calculated rejection was within an error goal (i.e., ±5%) from the experimental observation. To extend the GCM for a reference RO membrane, ESPA2-LD, 14 additional organics were included from the literature to calibrate nitrogen-containing functional groups of nitrosamine, nitriles, and amide compounds. Overall, 49 organics (72% of 68 compounds) from calibration and 7 compounds (87.5% of 8 compounds) from prediction were within the error goal.
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