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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 361 records · Page 20
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

AlphaFold predictions are valuable hypotheses and accelerate but do not replace experimental structure determination

Abstract Artificial intelligence-based protein structure prediction methods such as AlphaFold have revolutionized structural biology. The accuracies of these predictions vary, however, and they do not take into account ligands, covalent modifications or other environmental factors. Here, we evaluate how well AlphaFold predictions can be expected to describe the structure of a protein by comparing predictions directly with experimental crystallographic maps. In many cases, AlphaFold predictions matched experimental maps remarkably closely. In other cases, even very high-confidence predictions differed from experimental maps on a global scale through distortion and domain orientation, and on a local scale in backbone and side-chain conformation. We suggest considering AlphaFold predictions as exceptionally useful hypotheses. We further suggest that it is important to consider the confidence in prediction when interpreting AlphaFold predictions and to carry out experimental structure determination to verify structural details, particularly those that involve interactions not included in the prediction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF‐Trap

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Designer membraneless organelles sequester native factors for control of cell behavior

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. Here we report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to ninety percent of a targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division, and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Relevance of the Unfolded Protein Response to Spaceflight-Induced Transcriptional Reprogramming in Arabidopsis

Plants are primary producers of food and oxygen on Earth and will likewise be indispensable to the establishment of large-scale sustainable ecosystems and human survival in space. To contribute to the understanding of how plants respond to spaceflight stress, we examined the significance of the unfolded protein response (UPR), a conserved signaling cascade that responds to a number of unfavorable environmental stresses, in the model plant Arabidopsis thaliana. To do so, we performed a large-scale comparative transcriptome profiling in wild type and various UPR-defective mutants during the SpaceX-CRS12 mission to the International Space Station. We established that orbital culture substantially alters the expression of hundreds of stress-related genes compared with ground control conditions. Although expression of those genes varied in the UPR mutants on the ground, it was largely similar across the genotypes in the spaceflight condition. Furthermore, our results have yielded new information on how plants respond to growth in orbit and support the hypothesis that spaceflight induces the activation of signaling pathways that compensate for the loss of UPR regulators in the control of downstream transcriptional regulatory networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Challenges in predicting protein-protein interactions of understudied viruses: Arenavirus-human interactions

Understanding protein-protein interactions (PPIs) between viruses and host organisms is crucial for uncovering infection mechanisms and identifying potential therapeutic targets. The ability to generalize PPI predictive models across understudied viruses presents a significant challenge. In this work, we use arenavirus-human PPIs to illustrate the difficulties associated with model generalization, which are compounded by a lack of both positive and negative data. We employ a Transfer Learning approach to investigate arenavirus-human PPIs by utilizing models trained on better-studied virus-human and human-human PPIs. Additionally, we curate and assess four types of negative sampling datasets to evaluate their impact on model performance. Despite the overall high accuracies (93–99 %) and AUPRC scores (0.8–0.9) appearing promising, further analysis indicates that these performance metrics can be misleading due to data leakage, data bias, and overfitting, especially concerning under-represented viral proteins. We reveal these gaps and assess the impact of data imbalance using standard k-fold cross-validation and Independent Blind Testing with a Balanced Dataset, resulting in a drop in accuracy below 50 %. We propose a viral protein-specific evaluation framework that categorizes viral proteins into majority and minority classes based on their representation in the dataset, enabling comparison of model performance across these groups using balanced accuracies. This framework offers a more robust evaluation of model generalizability, addressing biases inherent in standard evaluation techniques and paving the way for more reliable PPI prediction models for understudied viruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dissection of Grass Cell Wall Lignin Formation and their Associated Dirigent Protein

This Final Report describes work underway to establish why monocot (e.g. grasses) lignins are more readily (bio)degradable than their woody gymnosperm, angiosperm, and fern counterparts. Our hypothesis is that monocot specific dirigent protein (DP) sub-families are wholly or partly involved in generating these more readily (bio)degradable lignins. Three approaches are underway: metabolomics and lignin analyses of stem and leaf tissues of the grass, Brachypodium; Crispr/Cas9 gene editing of monocot specific DP sub-families in this species, and recombinant protein expression of Brachypodium’s 44 DPs. Metabolomic and lignin analyses were completed for 6 and 8 week old Brachypodium in order to establish the molecular diversity of its largely phenolic metabolites: Crispr/Cas9 gene editing has also been done on specific DPs known to be expressed in lignifying stems, and T 2 generation plant lines are now being generated, prior to establishing the effects on lignin composition and content; in the recombinant DP work (which addresses establishing the biochemical function of the monocot specific DPs and other DPOs as needed), all 44 constructs have been generated (in collaboration with DOE JGI) and are being systematically expressed in either E. coli or yeast (Pichia sp.) for future studies. Future work will be directed towards bringing the current studies to completion, and in establishing the biochemical basis as to how these more readily (bio)degradable lignins are generated/assembled.

(bio)degradability↗
DOE OSTI2024

On the engulfment of antifreeze proteins by ice

Antifreeze proteins (AFPs) are remarkable biomolecules that suppress ice formation at trace concentrations. To inhibit ice growth, AFPs must not only bind to ice crystals, but also resist engulfment by ice. The highest supercooling, ΔT*, for which AFPs are able to resist engulfment is widely believed to scale as the inverse of the separation, L, between bound AFPs, whereas its dependence on the molecular characteristics of the AFP remains poorly understood. By using specialized molecular simulations and interfacial thermodynamics, here, we show that in contrast with conventional wisdom, ΔT* scales as L –2 and not as L –1 . We further show that ΔT* is proportional to AFP size and that diverse naturally occurring AFPs are optimal at resisting engulfment by ice. By facilitating the development of AFP structure–function relationships, we hope that our findings will pave the way for the rational design of AFPs.

Science & Technology - Other Topics↗
DOE OSTI2021

APS Science 2020 (Vol. 1)

The U.S. Department of Energy's Advanced Photon Source (APS) is one of the world’s most productive x-ray light source facilities. Each year, the APS provides high-brightness x-ray beams to a diverse community of more than 5,000 researchers in materials science, chemistry, condensed matter physics, the life and environmental sciences, and applied research. Researchers using the APS produce over 2,000 publications each year detailing impactful discoveries, and solve more vital biological protein structures than users of any other x-ray light source research facility. APS x-rays are ideally suited for explorations of materials and biological structures; elemental distribution; chemical, magnetic, electronic states; and a wide range of technologically important engineering systems from batteries to fuel injector sprays, all of which are the foundations of our nation’s economic, technological, and physical well-being. The APS occupies an 80-acre site on the Argonne campus, about 25 miles from downtown Chicago, Illinois. It shares a site with the Center for Nanoscale Materials and the Advanced Protein Characterization Facility.

25 ENERGY STORAGE↗
DOE OSTI2023

Quantitative differentiation of benign and misfolded glaucoma-causing myocilin variants on the basis of protein thermal stability

Accurate predictions of the pathogenicity of mutations associated with genetic diseases are key to the success of precision medicine. Inherited missense mutations in the myocilin (MYOC) gene, within its olfactomedin (OLF) domain, constitute the strongest genetic link to primary open-angle glaucoma via a toxic gain of function, and thus MYOC is an attractive precision-medicine target. However, not all mutations in MYOC cause glaucoma, and common variants are expected to be neutral polymorphisms. The Genome Aggregation Database (gnomAD) lists ~100 missense variants documented within OLF, all of which are relatively rare (allele frequency <0.001%) and nearly all are of unknown pathogenicity. To distinguish disease-causing OLF variants from benign OLF variants, we first characterized the most prevalent population-based variants using a suite of cellular and biophysical assays, and identified two variants with features of aggregation-prone familial disease variants. Next, we considered all available biochemical and clinical data to demonstrate that pathogenic and benign variants can be differentiated statistically based on a single metric: the thermal stability of OLF. Our results motivate genotyping MYOC in patients for clinical monitoring of this widespread, painless and irreversible ocular disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca 2+ Responsiveness

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca 2+ -controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca 2+ -dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein–protein interaction and Ca 2+ -binding motifs. Here we selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca 2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast ( Saccharomyces cerevisiae ) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca 2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca 2+ -controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca 2+ -dependent contractility.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Agave REVEILLE1 regulates the onset and release of seasonal dormancy in Populus

Abstract Deciduous woody plants like poplar (Populus spp.) have seasonal bud dormancy. It has been challenging to simultaneously delay the onset of bud dormancy in the fall and advance bud break in the spring, as bud dormancy, and bud break were thought to be controlled by different genetic factors. Here, we demonstrate that heterologous expression of the REVEILLE1 gene (named AaRVE1) from Agave (Agave americana) not only delays the onset of bud dormancy but also accelerates bud break in poplar in field trials. AaRVE1 heterologous expression increases poplar biomass yield by 166% in the greenhouse. Furthermore, we reveal that heterologous expression of AaRVE1 increases cytokinin contents, represses multiple dormancy-related genes, and up-regulates bud break-related genes, and that AaRVE1 functions as a transcriptional repressor and regulates the activity of the DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) promoter. Our findings demonstrate that AaRVE1 appears to function as a regulator of bud dormancy and bud break, which has important implications for extending the growing season of deciduous trees in frost-free temperate and subtropical regions to increase crop yield.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus

The control of virulence gene regulator (CovR), also called caspsule synthesis regulator (CsrR), is critical to how the major human pathogen group A Streptococcus fine-tunes virulence factor production. CovR phosphorylation (CovR~P) levels are determined by its cognate sensor kinase CovS, and functional abrogating mutations in CovS can occur in invasive GAS isolates leading to hypervirulence. Presently, the mechanism of CovR-DNA binding specificity is unclear, and the impact of CovS inactivation on global CovR binding has not been assessed. Thus, we performed CovR chromatin immunoprecipitation sequencing (ChIP-seq) analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and MGAS2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed virulence factor encoding genes could be grouped dependent on how CovR~P dependent variation in DNA binding correlated with gene transcript levels. Taken together, the data show that CovR repression of virulence factor encoding genes is primarily direct in nature, involves binding to a newly-identified DNA binding motif, and is relieved by CovS inactivation. These data provide new mechanistic insights into one of the most important bacterial virulence regulators and allow for subsequent focused investigations into how CovR-DNA interaction at directly controlled promoters impacts GAS pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

The protein structurome of Orthornavirae and its dark matter

Metatranscriptomics is uncovering more and more diverse families of viruses with RNA genomes comprising the viral kingdom Orthornavirae in the realm Riboviria. Thorough protein annotation and comparison are essential to get insights into the functions of viral proteins and virus evolution. In addition to sequence- and hmm profile-based methods, protein structure comparison adds a powerful tool to uncover protein functions and relationships. We constructed an Orthornavirae “structurome” consisting of already annotated as well as unannotated (“dark matter”) proteins and domains encoded in viral genomes. We used protein structure modeling and similarity searches to illuminate the remaining dark matter in hundreds of thousands of orthornavirus genomes. The vast majority of the dark matter domains showed either “generic” folds, such as single α-helices, or no high confidence structure predictions. Nevertheless, a variety of lineage-specific globular domains that were new either to orthornaviruses in general or to particular virus families were identified within the proteomic dark matter of orthornaviruses, including several predicted nucleic acid-binding domains and nucleases. In addition, we identified a case of exaptation of a cellular nucleoside monophosphate kinase as an RNA-binding protein in several virus families. Notwithstanding the continuing discovery of numerous orthornaviruses, it appears that all the protein domains conserved in large groups of viruses have already been identified. The rest of the viral proteome seems to be dominated by poorly structured domains including intrinsically disordered ones that likely mediate specific virus-host interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known. Here, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources. In this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

59 BASIC BIOLOGICAL SCIENCES↗