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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.

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At least 559 records · Page 31
All sourcesNASA NTRSDOE OSTI
DOE OSTI2020

Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO 2 -concentrating mechanism by facilitating high CO 2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein–protein interactions that drive the assembly process. In this work, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

γ-Aminobutyric acid modulates terpene biosynthesis through the ATG8a-mediated pathway

Terpenes play crucial roles in plant growth, development, and stress responses. The biosynthesis of terpenes is influenced by abiotic stress factors, such as drought, temperature, or light. Here, however, the molecular network underlying how terpenes are regulated in response to environmental stimuli remains largely unknown. Here, we identified the autophagy protein SgATG8a as a key mediator of GABA-regulated terpene production and drought tolerance in Sindora glabra. SgATG8a, evolutionarily related to the animal GABA receptor-associated protein (GABARAP) subfamily, localizes in both the nucleus and cytoplasm. Exogenous GABA treatment not only increased the expression level of terpene synthase genes (SgTPSs) but also led to enhanced accumulation of six main terpene components in Sindora glabra. In addition, GABA alleviated the photosynthesis damage and enhanced leaf biomass under drought conditions. Consistently, overexpression of SgATG8a in Arabidopsis increased terpene synthase gene (SgTPS) expression, leading to the enhanced production of four major terpenes and improved the tolerance of transgenic plants to drought stress by regulating reactive oxygen species (ROS) scavenging systems. Moreover, the transcription factors SgWRKY13 and SgERF4 were identified as interacting partners of SgATG8a, activating SgTPS3 expression. Lectin receptor-like kinase (LecRK1) is involved in the GABA-mediated pathway by interacting with the SgWRKY13/SgERF4-SgATG8a proteins, and the LecRK1-SgWRKY13/SgERF4 phosphorylation module fine-tunes the transcription of the downstream SgTPS3 gene. Taken together, these findings reveal a novel role for GABA in regulating terpene biosynthesis and drought tolerance, providing insights into the molecular mechanism underlying GABA-mediated terpene production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A minimal SufB 2 C 2 complex functions as a [4Fe-4S] cluster scaffold in methanogenic archaea

Iron-sulfur clusters are essential cofactors in all domains of life, yet their biogenesis in obligately anaerobic archaea remains poorly understood. Here, we characterized the minimal two-protein SUF system in methanogenic archaea, composed solely of SufB and SufC. Using Methanococcus maripaludis as a model, we demonstrate that the SUF proteins from its native host form a stable SufB 2 C 2 heterotetramer that binds a [4Fe-4S] cluster via three conserved cysteines in SufC. Mutations of conserved cysteine and histidine residues of SufB do not impair cluster binding. The complex interacts with the SAM-containing methanogenesis marker protein 10 (MmpX), suggesting direct Fe-S cluster transfer from SufB 2 C 2 to target proteins. Mutational analysis of Methanothermococcus thermolithotrophicus proteins confirmed that SufC is the primary cluster-binding component, while SufB enhances ATPase and cluster transfer activities. Evolutionary comparisons suggest that this two-protein SUF system represents an ancestral form of Fe-S cluster biogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Investigating and Optimizing the Lysate-Based Expression of Nonribosomal Peptide Synthetases Using a Reporter System

Lysate-based cell-free expression (CFE) systems are accessible platforms for expressing proteins that are difficult to synthesize in vivo, such as nonribosomal peptide synthetases (NRPSs). NRPSs are large (>100 kDa), modular enzyme complexes that synthesize bioactive peptide natural products. This synthetic process is analogous to transcription/translation (TX/TL) in lysates, resulting in potential resource competition between NRPS expression and NRPS activity in cell-free environments. Moreover, CFE conditions depend on the size and structure of the protein. Here, a reporter system for rapidly investigating and optimizing reaction environments for NRPS CFE is described. This strategy is demonstrated in E. coli lysate reactions using blue pigment synthetase A (BpsA), a model NRPS, carrying a C-terminal tetracysteine (TC) tag which forms a fluorescent complex with the biarsenical dye, FlAsH. A colorimetric assay was adapted for lysate reactions to detect the blue pigment product, indigoidine, of cell-free expressed BpsA-TC, confirming that the tagged enzyme is catalytically active. An optimized protocol for end point TC/FlAsH complex measurements in reactions enables quick comparisons of full-length BpsA-TC expressed under different reaction conditions, defining unique requirements for NRPS expression that are related to the protein’s catalytic activity and size. Importantly, these protein-dependent CFE conditions enable higher indigoidine titer and improve the expression of other monomodular NRPSs. Notably, these conditions differ from those used for the expression of superfolder GFP (sfGFP), a common reporter for optimizing lysate-based CFE systems, indicating the necessity for tailored reporters to optimize expression for specific enzyme classes. In conclusion, the reporter system is anticipated to advance lysate-based CFE systems for complex enzyme synthesis, enabling natural product discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Hallucination of closed repeat proteins containing central pockets

In pseudocyclic proteins, such as TIM barrels, β barrels, and some helical transmembrane channels, a single subunit is repeated in a cyclic pattern, giving rise to a central cavity that can serve as a pocket for ligand binding or enzymatic activity. Inspired by these proteins, we devised a deep-learning-based approach to broadly exploring the space of closed repeat proteins starting from only a specification of the repeat number and length. Biophysical data for 38 structurally diverse pseudocyclic designs produced in Escherichia coli are consistent with the design models, and the three crystal structures we were able to obtain are very close to the designed structures. Docking studies suggest the diversity of folds and central pockets provide effective starting points for designing small-molecule binders and enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Biomolecular complex viewed by dynamic nuclear polarization solid-state NMR spectroscopy

Solid-state nuclear magnetic resonance (ssNMR) is an indispensable tool for elucidating the structure and dynamics of insoluble and non-crystalline biomolecules. The recent advances in the sensitivity-enhancing technique magic-angle spinning dynamic nuclear polarization (MAS-DNP) have substantially expanded the territory of ssNMR investigations and enabled the detection of polymer interfaces in a cellular environment. This article highlights the emerging MAS-DNP approaches and their applications to the analysis of biomolecular composites and intact cells to determine the folding pathway and ligand binding of proteins, the structural polymorphism of low-populated biopolymers, as well as the physical interactions between carbohydrates, proteins, and lignin. Furthermore, these structural features provide an atomic-level understanding of many cellular processes, promoting the development of better biomaterials and inhibitors. It is anticipated that the capabilities of MAS-DNP in biomolecular and biomaterial research will be further enlarged by the rapid development of instrumentation and methodology.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Pose Classification Using Three-Dimensional Atomic Structure-Based Neural Networks Applied to Ion Channel–Ligand Docking

The identification of promising lead compounds showing pharmacological activities toward a biological target is essential in early stage drug discovery. With the recent increase in available small-molecule databases, virtual high-throughput screening using physics-based molecular docking has emerged as an essential tool in assisting fast and cost-efficient lead discovery and optimization. However, the best scored docking poses are often suboptimal, resulting in incorrect screening and chemical property calculation. We address the pose classification problem by leveraging data-driven machine learning approaches to identify correct docking poses from AutoDock Vina and Glide screens. To enable effective classification of docking poses, we present two convolutional neural network approaches: a three-dimensional convolutional neural network (3D-CNN) and an attention-based point cloud network (PCN) trained on the PDBbind refined set. We demonstrate the effectiveness of our proposed classifiers on multiple evaluation data sets including the standard PDBbind CASF-2016 benchmark data set and various compound libraries with structurally different protein targets including an ion channel data set extracted from Protein Data Bank (PDB) and an in-house KCa3.1 inhibitor data set. Our experiments show that excluding false positive docking poses using the proposed classifiers improves virtual high-throughput screening to identify novel molecules against each target protein compared to the initial screen based on the docking scores.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

SARS-CoV2 Protein-Ligand Simulation Dataset: Layer 2 (Lowest Temperature Extracted Protein Coordinate Trajectories)

Protein-only trajectories of the lowest-temperature window (310K) extracted from temperature replica-exchange molecular dynamics simulations of 23 different SARS CoV-2 systems, including S Protein ACE2-receptor binding-domain, MPro, PLPro, NSP3 ADRP (X-domain/macrodomain/phosphatase), NSP15 (endoribonuclease), NSP9, NSP10, NSP16, and N-protein N-terminus. Systems were prepared with charmm-gui and simulated using the GROMACS simulation software suite. Trajectories are provided in compressed dcd format with accompanying coordinate/topology files in pdb and psf formats. This data supplements the data release, DOI: 10.13139/OLCF/1650650 ('SARS-CoV2 Protein-Ligand Simulation Dataset: Layer 1 (Simulation Initial Conditions and Parameters)')

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1 C ) regions to GP metastability. Specific stalk and HR1 C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1 C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Lipocalin Blc is a potential heme-binding protein

Lipocalins are a superfamily of functionally diverse proteins defined by a well-conserved tertiary structure despite variation in sequence. Lipocalins bind and transport small hydrophobic molecules in organisms of all kingdoms. However, there is still uncertainty regarding the function of some members of the family, including bacterial lipocalin Blc from Escherichia coli. Here, we present evidence that lipocalin Blc may be involved in heme binding, trans-periplasmic transport, or heme storage. This conclusion is supported by a cocrystal structure, mass-spectrometric data, absorption titration, and in silico analysis. Binding of heme is observed at low micromolar range with one-to-one ligand-to-protein stoichiometry. However, the absence of classical coordination to the iron atom leaves the possibility that the primary ligand of Blc is another tetrapyrrole.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification and expression of small multidrug resistance transporters in early‐branching anaerobic fungi

Abstract Membrane‐embedded transporters impart essential functions to cells as they mediate sensing and the uptake and extrusion of nutrients, waste products, and effector molecules. Promiscuous multidrug exporters are implicated in resistance to drugs and antibiotics and are highly relevant for microbial engineers who seek to enhance the tolerance of cell factory strains to hydrophobic bioproducts. Here, we report on the identification of small multidrug resistance (SMR) transporters in early‐branching anaerobic fungi (Neocallimastigomycetes). The SMR class of transporters is commonly found in bacteria but has not previously been reported in eukaryotes. In this study, we show that SMR transporters from anaerobic fungi can be produced heterologously in the model yeast Saccharomyces cerevisiae , demonstrating the potential of these proteins as targets for further characterization. The discovery of these novel anaerobic fungal SMR transporters offers a promising path forward to enhance bioproduction from engineered microbial strains.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

iPNHOT: a knowledge-based approach for identifying protein-nucleic acid interaction hot spots

The interaction between proteins and nucleic acids plays pivotal roles in various biological processes such as transcription, translation, and gene regulation. Hot spots are a small set of residues that contribute most to the binding affinity of a protein-nucleic acid interaction. Compared to the extensive studies of the hot spots on protein-protein interfaces, the hot spot residues within protein-nucleic acids interfaces remain less well-studied, in part because mutagenesis data for protein-nucleic acids interaction are not as abundant as that for protein-protein interactions. In this study, we built a new computational model, iPNHOT, to effectively predict hot spot residues on protein-nucleic acids interfaces. One training data set and an independent test set were collected from dbAMEPNI and some recent literature, respectively. To build our model, we generated 97 different sequential and structural features and used a two-step strategy to select the relevant features. The final model was built based only on 7 features using a support vector machine (SVM). The features include two unique features such as ΔSASsa 1/2 and esp3, which are newly proposed in this study. Based on the cross validation results, our model gave F1 score and AUROC as 0.725 and 0.807 on the subset collected from ProNIT, respectively, compared to 0.407 and 0.670 of mCSM-NA, a state-of-the art model to predict the thermodynamic effects of protein-nucleic acid interaction. The iPNHOT model was further tested on the independent test set, which showed that our model outperformed other methods. Here, by collecting data from a recently published database dbAMEPNI, we proposed a new model, iPNHOT, to predict hotspots on both protein-DNA and protein-RNA interfaces. The results show that our model outperforms the existing state-of-art models. Our model is available for users through a webserver: http://zhulab.ahu.edu.cn/iPNHOT/.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Next-generation yeast-two-hybrid analysis with Y2H-SCORES identifies novel interactors of the MLA immune receptor

Protein-protein interaction networks are one of the most effective representations of cellular behavior. In order to build these models, high-throughput techniques are required. Next-generation interaction screening (NGIS) protocols that combine yeast two-hybrid (Y2H) with deep sequencing are promising approaches to generate interactome networks in any organism. However, challenges remain to mining reliable information from these screens and thus, limit its broader implementation. Here, we present a computational framework, designated Y2H-SCORES, for analyzing high-throughput Y2H screens. Y2H-SCORES considers key aspects of NGIS experimental design and important characteristics of the resulting data that distinguish it from RNA-seq expression datasets. Three quantitative ranking scores were implemented to identify interacting partners, comprising: 1) significant enrichment under selection for positive interactions, 2) degree of interaction specificity among multi-bait comparisons, and 3) selection of in-frame interactors. Using simulation and an empirical dataset, we provide a quantitative assessment to predict interacting partners under a wide range of experimental scenarios, facilitating independent confirmation by one-to-one bait-prey tests. Simulation of Y2H-NGIS enabled us to identify conditions that maximize detection of true interactors, which can be achieved with protocols such as prey library normalization, maintenance of larger culture volumes and replication of experimental treatments. Y2H-SCORES can be implemented in different yeast-based interaction screenings, with an equivalent or superior performance than existing methods. Proof-of-concept was demonstrated by discovery and validation of novel interactions between the barley nucleotide-binding leucine-rich repeat (NLR) immune receptor MLA6, and fourteen proteins, including those that function in signaling, transcriptional regulation, and intracellular trafficking.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe 2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe 2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein–protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen–deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein–protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA–NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Sugar phosphate activation of the stress sensor eIF2B

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Protein model quality assessment using rotation–equivariant transformations on point clouds

Machine learning research concerning protein structure has seen a surge in popularity over the last years with promising advances for basic science and drug discovery. Working with macromolecular structure in a machine learning context requires an adequate numerical representation, and researchers have extensively studied representations such as graphs, discretized 3D grids, and distance maps. As part of CASP14, we explored a new and conceptually simple representation in a blind experiment: atoms as points in 3D, each with associated features. These features—initially just the basic element type of each atom—are updated through a series of neural network layers featuring rotation-equivariant convolutions. Starting from all atoms, we further aggregate information at the level of alpha carbons before making a prediction at the level of the entire protein structure. We find that this approach yields competitive results in protein model quality assessment despite its simplicity and despite the fact that it incorporates minimal prior information and is trained on relatively little data. As a result, its performance and generality are particularly noteworthy in an era where highly complex, customized machine learning methods such as AlphaFold 2 have come to dominate protein structure prediction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Sequence, structure prediction, and epitope analysis of the polymorphic membrane protein family in Chlamydia trachomatis

The polymorphic membrane proteins (Pmps) are a family of autotransporters that play an important role in infection, adhesion and immunity in Chlamydia trachomatis. Here we show that the characteristic GGA(I,L,V) and FxxN tetrapeptide repeats fit into a larger repeat sequence, which correspond to the coils of a large beta-helical domain in high quality structure predictions. Analysis of the protein using structure prediction algorithms provided novel insight to the chlamydial Pmp family of proteins. While the tetrapeptide motifs themselves are predicted to play a structural role in folding and close stacking of the beta-helical backbone of the passenger domain, we found many of the interesting features of Pmps are localized to the side loops jutting out from the beta helix including protease cleavage, host cell adhesion, and B-cell epitopes; while T-cell epitopes are predominantly found in the beta-helix itself. This analysis more accurately defines the Pmp family of Chlamydia and may better inform rational vaccine design and functional studies.

59 BASIC BIOLOGICAL SCIENCES↗