Search NASA⌕ Search

SEARCH · Search NASA

Results for “Protein engineering”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 271 records · Page 15

Sequence-based generative AI design of versatile tryptophan synthases

Enzymes are powerful and sustainable catalysts, but their widespread application is limited by the difficulty of identifying functional starting points for optimization, creating a major bottleneck in early- stage biocatalyst discovery. Designing libraries of such starting enzymes remains particularly challenging. Here, we use the GenSLM protein language model to generate novel β-subunit of tryptophan synthase (TrpB) enzymes that express in Escherichia coli and are both stable and catalytically active. Many generated TrpBs also display significant substrate promiscuity, outperforming their natural counterparts on non-native substrates. Some even surpass laboratory-evolved TrpBs. Comparison of the most-active and most-promiscuous generated TrpB to its closest natural homolog confirms that the enhanced versatility is absent from the natural enzyme, highlighting the creative potential of generative models. These results demonstrate that the generated TrpBs not only preserve natural structure and function but also acquire non-natural properties, establishing generative models as powerful tools for biocatalyst discovery and engineering.

biocatalysis↗

Developing affordable and efficient heating devices for enhanced live cell imaging in confocal microscopy

Temperature control is crucial for live cell imaging, particularly in studies involving plant responses to high ambient temperatures and thermal stress. This study presents the design, development, and testing of two cost-effective heating devices tailored for confocal microscopy applications: an aluminum heat plate and a wireless mini-heater. The aluminum heat plate, engineered to integrate seamlessly with the standard 160 mm × 110 mm microscope stage, supports temperatures up to 36°C, suitable for studies in the range of non-stressful warm temperatures (e.g., 25-27°C forArabidopsis thaliana) and moderate heat stress (e.g., 30-36°C forA. thaliana). We also developed a wireless mini-heater that offers rapid, precise heating directly at the sample slide, with a temperature increase rate over 30 times faster than the heat plate. The wireless heater effectively maintained target temperatures up to 50°C, ideal for investigating severe heat stress and heat shock responses in plants. Both devices performed well in controlled studies, including the real-time analysis of heat shock protein accumulation and stress granule formation inA. thaliana. Our designs are effective and affordable, with total construction costs lower than $300. This accessibility makes them particularly valuable for small laboratories with limited funding. Future improvements could include enhanced heat uniformity, humidity control to mitigate evaporation, and more robust thermal management to minimize focus drift during extended imaging sessions. These modifications would further solidify the utility of our heating devices in live cell imaging, offering researchers reliable, budget-friendly tools for exploring plant thermal biology.

Plant Sciences↗

Final Report for FE0032098: Improving the cost-effectiveness of algal CO2 utilization by synergistic integration with power plant and wastewater treatment operations

The overall goal of this project was to demonstrate an engineering-scale open raceway pond algae cultivation system (approximately 180 m²), including the integration of technologies that utilized carbon dioxide (CO₂) from a coal-fired power plant and wastewater-derived nutrient inputs for cost-effective and environmentally friendly biomass production. The key advantages associated with the innovative algae cultivation system and its integration with wastewater treatment functions, as described herein, had been demonstrated in previous bench- and pilot-scale work by the project team partners. This project combined those approaches to maximize practical benefits and available synergies, resulting in a significant reduction in the net cost of producing algal biomass products. The primary target algal species for the project was Spirulina, which served as a high-protein content ingredient for food and animal feed. Spirulina was selected because it had already been approved by the FDA, was in use as a food ingredient, and commanded prices of up to $30/kg. It had a typical protein content of 50–75%, comparable to other high-protein concentrates, featured high digestibility without requiring pretreatment, and offered a high conversion ratio in animal feed applications. In addition, Spirulina had a relatively high content of the blue pigment phycocyanin, which could be extracted as a high-value co-product prior to using the remaining biomass for nutritional purposes.

Schideman, Lance [University of Illinois]↗

Biotransformation of Pesticides across Biological Systems: Molecular Mechanisms, Omics Insights, and Biotechnological Advances for Environmental Sustainability

The widespread application of pesticides such as organophosphates, organochlorides, and triazines in modern agriculture has led to their notable presence in soils, water bodies, and food chains, raising concerns about persistence, bioaccumulation, and adverse effects on nontarget organisms. Biotransformation, the enzymatic transformation of xenobiotic compounds by microorganisms, plants, and animals, plays a pivotal role in the degradation and detoxification of these chemicals. This review provides a comprehensive examination of the mechanisms, key enzyme classes (e.g., hydrolases, oxidoreductases, transferases), and environmental factors influencing pesticide biotransformation across different biological systems. Recent advances in omics technologies have revolutionized the understanding of microbial and plant metabolism, while synthetic biology offers opportunities for engineering enhanced degradation capabilities. The environmental fate of transformation products is also discussed, together with a critical analysis of challenges, unresolved questions, and future research directions, offering a holistic perspective on pesticide biotransformation as a key process for mitigating chemical pollution.

Biotransformation↗

Integrative mapping reveals molecular features underlying the mechanism of nucleocytoplasmic transport

Nuclear pore complexes (NPCs) enable rapid, selective, and robust nucleocytoplasmic transport. To explain how transport emerges from the system components and their interactions, we used experimental data and theoretical information to construct an integrative Brownian dynamics model of transport through an NPC, coupled to a kinetic model of transport in the cell. The model recapitulates key aspects of transport for a wide range of molecular cargoes, including preribosomes and viral capsids. Our model quantifies how flexible phenylalanine-glycine (FG) repeat proteins create an entropic barrier to passive diffusion and how this barrier is selectively lowered in facilitated diffusion by the many transient interactions of nuclear transport receptors with the FG repeats. Selective transport is enhanced by “fuzzy” multivalent interactions, redundant FG repeat mass, coupling to the energy-dependent RanGTP concentration gradient, and exponential dependence of transport kinetics on the transport barrier. Our model will facilitate rational modulation of the NPC and its artificial mimics.

Raveh, Barak [School of Computer Science and Engin↗

Unfolding of the Villin Headpiece Domain: Revealing Structural Heterogeneity with Time‐Resolved X‐Ray Solution Scattering and Markov State Modeling

Understanding protein folding pathways is crucial to deciphering the principles of protein structure and function. Here, the unfolding dynamics of the 35‐residue villin headpiece (HP35) and a norleucine‐substituted variant (2F4K) using a combination of experimental and computational techniques is investigated. Time‐resolved X‐ray solution scattering coupled with equilibrium molecular dynamics simulations and Markov state modeling reveals distinct unfolding mechanisms between the two variants: HP35 and 2F4K. Specifically, HP35 exhibits a two‐state unfolding process, whereas an intermediate state is identified for the 2F4K mutant. A Markov state model constructed from simulations is used to map atomic‐level transitions to experimental observations, providing insights into the role of sequence variations in modulating folding pathways. The findings underscore the importance of integrating experimental and computational approaches to unravel protein unfolding mechanisms between heterogenous structural ensembles.

Nijhawan, Adam K. [Department of Chemistry Northwe↗

Crystal structure and catalytic mechanism of drimenol synthase, an unusual bifunctional terpene cyclase–phosphatase

Drimenol synthase fromAquimarina spongiae(AsDMS) is a highly unusual chimera that integrates two distinct, sequential isoprenoid processing activities within a single polypeptide chain. AsDMS catalyzes the class II cyclization of farnesyl diphosphate (FPP) to form drimenyl diphosphate, which then undergoes enzyme-catalyzed hydrolysis to yield drimenol, a bioactive sesquiterpene alcohol with antifungal and anticancer properties. Here, we report the X-ray crystal structures of AsDMS and its complex with a sesquiterpene thiol. The AsDMS structure exhibits a didomain architecture consisting of a terpene cyclase β domain and a haloacid dehalogenase-like phosphatase domain, with two distinct active sites located on opposite sides of the protein. Mechanistic studies show that dephosphorylation of the drimenyl diphosphate intermediate proceeds through stepwise hydrolysis such that two equivalents of inorganic phosphate rather than inorganic pyrophosphate are coproducts of the reaction sequence. When the AsDMS reaction is performed in H 2 18 O, 18 O is not incorporated into drimenol, indicating that the hydroxyl oxygen of drimenol originates from the prenyl oxygen of FPP rather than a water molecule from bulk solution. These results correct a mechanistic proposal previously advanced by another group. Surprisingly, AsDMS exhibits substrate promiscuity, catalyzing the conversion of the slowly reactive substrate mimic farnesyl-S-thiolodiphosphate into cyclic and linear sesquiterpene products. Structural and mechanistic insights gained from AsDMS illustrate the functional diversity of terpene biosynthetic enzymes and provide a foundation for engineering “designer cyclase” assemblies capable of generating a wide variety of terpenoid products.

Science & Technology - Other Topics↗

Hyporheic zone, river, and groundwater metagenome resolved genomes and rpS3 genes in East River Watershed, Colorado USA Summer 2020, 2021

Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from water filter collected across 8 locations along the East River Watershed, CO, and 1 nearby groundwater well. The purpose was to look for connectivity and similarities across the network and to see the impact of the groundwater. As a part of Lawrence Berkeley National Laboratory (LBNL) Watershed Science Focus Area (SFA), we assessed community composition and strain similarities between the sites and we also compared it to previous metagenomic studies within the watershed looking at floodplain (Matheus Carnevali et al. 2021) and hillslope (Lavy et al. 2019) microbiomes. Here we present metagenome assembled genomes (MAGs) for the bacterial and archaeal communities from filters across 8 locations during August 2020 and July 2021. This resulted in 32 samples. The groundwater sample was sequenced at UC Berkley's QB3. The other 31 samples were sequenced at University of Maryland. Metagenomes were assembled using four autobinners and the best bins were selected using dasTool. The genomes were dereplicated at 95% with dRep and the subset of winning genomes were manually curated based on visual inspection of taxonomic profile, GC content, coverage, and a set of 51 bacterial single copy genes (BSCG), and 38 archaeal signal copy genes (ASCG). The dataset includes a zip file of 311 genomes (HZ_River_SW_MAGS_Dereplicated_95.zip). The dataset additionally includes a zipped file of ribosomal protein small subunit 3 (rpS3) proteins from the hyporheic zone and river data (rpS3_Proteins_HZ_River.zip), a metadata file used to register associated samples with IGSNs (International Generic Sample Numbers) (samples.csv), a location metadata file (locations.csv). This work was supported by the Watershed Function Science Focus Area at Lawrence Berkeley National Laboratory funded by the US Department of Energy, Office of Science, Biological and Environmental Research under Contract No. DE-AC02-05CH11231.

DNA↗

Cryo-EM Visualization of Intermolecular π-Electron Interactions within π-Conjugated Peptidic Supramolecular Polymers

The self-assembly of “π-peptides” – molecules with π-electron cores substituted with two or more oligopeptide chains – brings organic electronic function into biologically relevant nanomaterials. π-Peptides assemble into fibrillar nanomaterials as driven by enthalpic peptide-based hydrogen bonding networks and pi-core-based quadrupolar interactions. A large body of spectroscopic, morphological and computational studies informs on the nature of the self-assembly process and the resulting nanostructures, but detailed structural information has remained elusive. Here, inspired by the recent use of cryogenic electron microscopy (cryo-EM) to provide high-resolution structures for synthetic peptide nanomaterials, we present here the use of cryo-EM to offer ca. 3 Å resolution of π-peptide nanomaterial assemblies, visualizing for the first time the nature of the intermolecular π-core electronic interactions responsible for energy transport through these supramolecular materials.

Group theory↗

Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production

Abstract The aviation industry’s growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multiomics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to 3 d earlier at 10 °C. However, these alterations led to a tradeoff between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.

Rasoul, Amira (ORCID:0009000714290175)↗

Plant Cell Wall Polysaccharide O-Acetyltransferases

Plant cell walls are largely composed of polysaccharide polymers, including cellulose, hemicelluloses (xyloglucan, xylan, mannan, and mixed-linkage β-1,3/1,4-glucan), and pectins. Among these cell wall polysaccharides, xyloglucan, xylan, mannan, and pectins are often O-acetylated, and polysaccharide O-acetylation plays important roles in cell wall assembly and disease resistance. Genetic and biochemical analyses have implicated the involvement of three groups of proteins in plant cell wall polysaccharide O-acetylation: trichome birefringence-like (TBL)/domain of unknown function 231 (DUF231), reduced wall acetylation (RWA), and altered xyloglucan 9 (AXY9). Although the exact roles of RWAs and AXY9 are yet to be identified, members of the TBL/DUF231 family have been found to be O-acetyltransferases responsible for the O-acetylation of xyloglucan, xylan, mannan, and pectins. Here, we provide a comprehensive overview of the occurrence of O-acetylated cell wall polysaccharides, the biochemical properties, structural features, and evolution of cell wall polysaccharide O-acetyltransferases, and the potential biotechnological applications of manipulations of cell wall polysaccharide acetylation. Further in-depth studies of the biochemical mechanisms of cell wall polysaccharide O-acetylation will not only enrich our understanding of cell wall biology, but also have important implications in engineering plants with increased disease resistance and reduced recalcitrance for biofuel production.

Plant Sciences↗

Data for Creating Yellow Seed Camelina sativa with Enhanced Oil Accumulation by CRISPR-Mediated Disruption of Transparent Testa 8

Camelina ( Camelina sativa L.), a hexaploid member of the Brassicaceae family, is an emerging oilseed crop being developed to meet the increasing demand for plant oils as biofuel feedstocks. In other Brassicas, high oil content can be associated with a yellow seed phenotype, which is unknown for camelina. We sought to create yellow seed camelina using CRISPR/Cas9 technology to disrupt its Transparent Testa 8 (TT8) transcription factor genes and to evaluate the resulting seed phenotype. We identified three TT8 genes, one in each of the three camelina subgenomes, and obtained independent CsTT8 lines containing frameshift edits. Disruption of TT8 caused seed coat colour to change from brown to yellow reflecting their reduced flavonoid accumulation of up to 44%, and the loss of a well-organized seed coat mucilage layer. Transcriptomic analysis of CsTT8-edited seeds revealed significantly increased expression of the lipid-related transcription factors LEC1, LEC2, FUS3, and WRI1 and their downstream fatty acid synthesis-related targets. These changes caused metabolic remodelling with increased fatty acid synthesis rates and corresponding increases in total fatty acid (TFA) accumulation from 32.4% to as high as 38.0% of seed weight, and TAG yield by more than 21% without significant changes in starch or protein levels compared to parental line. These data highlight the effectiveness of CRISPR in creating novel enhanced-oil germplasm in camelina. The resulting lines may directly contribute to future net-zero carbon energy production or be combined with other traits to produce desired lipid-derived bioproducts at high yields.

Biofuels↗

Environmental Contributions to Proton Sharing in Protein Low-Barrier Hydrogen Bonds

Hydrogen bonds (H-bonds) are central to biomolecular structure and dynamics. Although H-bonds are typically characterized by well-defined proton positions, proton delocalization has been proposed to play a role in facilitating enzyme catalysis and allostery in some systems. Experimentally locating protons is difficult, hampering the study of proton mobility in H-bonds. We used neutron crystallography, atomic resolution X-ray bond length analysis, and large quantum mechanics/molecular mechanics-Born–Oppenheimer molecular dynamics (QM/MM-BOMD) simulations to comprehensively characterize the shared proton/deuteron in a Glu–Asp low-barrier hydrogen bond (LBHB) in the bacterial protein YajL that is a conventional H-bond in the homologous disease-associated human protein DJ-1. X-ray bond length analysis of protiated and perdeuterated DJ-1 and YajL shows no significant effect of deuteron substitution on these carboxylic acid-carboxylate H-bonds but does reveal an effect at the active site glutamic acid near a cysteine thiolate. Residues in an H-bonded network that might favor LBHB formation in YajL were interrogated by the mutation of homologous residues in DJ-1. A distal DJ-1 substitution increases proton delocalization in the Glu–Asp H-bond, demonstrating that mutations within extended H-bond networks can modulate proton transfer barriers in carboxylic acid-carboxylate H-bonds. In addition, proton mobility in the H-bond is correlated with dimer-spanning motions in the QM/MM-BOMD simulations of YajL and DJ-1. Our results show that proton delocalization can be tuned using combined bioinformatic, structural, and computational information, opening the possibility of using engineered proton delocalization as a probe of H-bonding environments and as a tool to test hypotheses about LBHB function.

Lin, Jiusheng [University of Nebraska, Lincoln, NE↗

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

Genome-scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling and pairing product formation with substrate utilization and can improve strain performance beyond levels typically accessible using traditional strain engineering approaches. However, sustainable feedstocks pose a challenge due to incomplete high-resolution metabolic data for non-canonical carbon sources required to curate GSMM and identify implementable designs. Here we address a four-gene deletion design in the Pseudomonas putida KT2440 strain for the lignin-derived non-sugar carbon source, p-coumarate (p-CA), that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product that is commonly used to colorimetrically quantify glutamine concentration. Through proteomics, promoter-variation, and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarase hydratase (FUM) enzyme activity in the citrate cycle and requires careful optimization of another fumarate hydratase protein (PP_0897) expression to achieve growth and production. A double sensitivity analysis also confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. Metabolic cross-feeding experiments were used to examine the impact of complete removal of the fumarase hydratase reaction and revealed an unanticipated nutrient requirement, suggesting additional functions for this enzyme. While a complete implementation of the design was achieved, this study highlights the challenge of completely inactivating metabolic reactions encoded by under-characterized proteins, especially in the context of multi-gene edits.

59 BASIC BIOLOGICAL SCIENCES↗

Next-Generation Sequencing Data from a CUT&RUN Study of R. toruloides IFO0880 Cse4 and Orc1 Binding Sites

Rhodotorula toruloides has been increasingly explored as a host for bioproduction of lipids, fatty acid derivatives and terpenoids. Various genetic tools have been developed, but neither a centromere nor an autonomously replicating sequence (ARS), both necessary elements for stable episomal plasmid maintenance, has yet been reported. In this study, cleavage under targets and release using nuclease (CUT&RUN), a method used for genome-wide mapping of DNA–protein interactions, was used to identify R. toruloides IFO0880 genomic regions associated with the centromeric histone H3 protein Cse4, a marker of centromeric DNA. Fifteen putative centromeres ranging from 8 to 19 kb in length were identified and analyzed, and four were tested for, but did not show, ARS activity. These centromeric sequences contained below average GC content, corresponded to transcriptional cold spots, were primarily nonrepetitive and shared some vestigial transposon-related sequences but otherwise did not show significant sequence conservation. Future efforts to identify an ARS in this yeast can utilize these centromeric DNA sequences to improve the stability of episomal plasmids derived from putative ARS elements.

Genome Engineering↗

Dual Enhancement of Thermostability and Activity of Xylanase through Computer-Aided Rational Design

In the realm of enzyme engineering, the dual enhancement of thermostability and activity remains a challenge. Herein, we employed a computer-aided approach integrating folding free energy calculations and evolutionary analysis to engineer Paecilomyces thermophila xylanase into a hyperthermophilic enzyme for application in the paper and pulp industry. Through the computational rational design, XynM9 with superior thermostability and enhanced activity was designed. Its optimal reaction temperature increases by 10 °C to 85 °C, its T m increases by 10 °C to 93 °C, and its half-life increases 11-fold to 5.8 h. Additionally, its catalytic efficiency improves by 57% to 3926 s –1 mM –1 . Molecular dynamics simulations revealed that XynM9 is stabilized by more hydrogen bonds and salt bridges than wild-type xylanase. The mutant’s narrower catalytic cleft enhances the substrate-binding affinity, thus improving the catalytic efficiency. In harsh conditions at 80 °C and pH 10, using XynM9 significantly reduced both hemicellulose and lignin, which makes it a good candidate for use in the paper and pulp process. Finally, our study presents an accurate and efficient strategy for the dual enhancement of enzyme properties, guiding further improvement of computational tools for protein stabilization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Torsional twist of the SARS ‐ CoV and SARS ‐ CoV ‐2 SUD ‐N and SUD ‐M domains

Abstract Coronavirus non‐structural protein 3 (nsp3) forms hexameric crowns of pores in the double membrane vesicle that houses the replication–transcription complex. Nsp3 in SARS‐like viruses has three unique domains absent in other coronavirus nsp3 proteins. Two of these, SUD‐N (Macrodomain 2) and SUD‐M (Macrodomain 3), form two lobes connected by a peptide linker and an interdomain disulfide bridge. We resolve the first complete x‐ray structure of SARS‐CoV SUD‐N/M as well as a mutant variant of SARS‐CoV‐2 SUD‐N/M modified to restore cysteines for interdomain disulfide bond naturally lost by evolution. Comparative analysis of all structures revealed SUD‐N and SUD‐M are not rigidly associated but rather have significant rotational flexibility. Phylogenetic analysis supports that the potential to form the disulfide bond is common across betacoronavirus isolates from many bat species and civets, but also one or both of the cysteines that form the disulfide bond are absent across isolates from bats and pangolins. The absence of these cysteines does not impact viral replication or protein translation.

Rosas‐Lemus, Monica [Department of Microbiology‐Im↗