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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 433 records · Page 24
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Tellurite Adsorption onto Bacterial Surfaces

Tellurium (Te) is an emerging contaminant and its chemical transformation in the environment is strongly influenced by microbial processes. In this study, we investigated the adsorption of tellurite [Te(IV), TeO 3 2- ] onto the common soil bacterium Bacillus subtilis. Thiol-blocking experiments were carried out to investigate the role of cell surface sulfhydryl sites in tellurite binding, and extended X-ray absorption fine structure (EXAFS) spectroscopy was performed to determine the chemical speciation of the adsorbed tellurite. The results indicate that tellurite reacts with sulfhydryl functional groups in the extracellular polymeric substances (EPS) produced by B. subtilis. Upon binding to sulfhydryl sites in the EPS, the Te changes from Te-O bonds to Te-S coordination. Further analysis of the surface-associated molecules shows that the EPS of B. subtilis contain proteins. Removal of the proteinaceous EPS dramatically decreases tellurite adsorption and the sulfhydryl surface site concentration. These findings indicate that sulfhydryl binding in EPS plays a key role in tellurite adsorption on bacterial surfaces.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Discovery of Small-Molecule Inhibitors of SARS-CoV-2 Proteins Using a Computational and Experimental Pipeline

A rapid response is necessary to contain emergent biological outbreaks before they can become pandemics. The novel coronavirus (SARS-CoV-2) that causes COVID-19 was first reported in December of 2019 in Wuhan, China and reached most corners of the globe in less than two months. In just over a year since the initial infections, COVID-19 infected almost 100 million people worldwide. Although similar to SARS-CoV and MERS-CoV, SARS-CoV-2 has resisted treatments that are effective against other coronaviruses. Crystal structures of two SARS-CoV-2 proteins, spike protein and main protease, have been reported and can serve as targets for studies in neutralizing this threat. We have employed molecular docking, molecular dynamics simulations, and machine learning to identify from a library of 26 million molecules possible candidate compounds that may attenuate or neutralize the effects of this virus. The viability of selected candidate compounds against SARS-CoV-2 was determined experimentally by biolayer interferometry and FRET-based activity protein assays along with virus-based assays. In the pseudovirus assay, imatinib and lapatinib had IC 50 values below 10 μM, while candesartan cilexetil had an IC 50 value of approximately 67 µM against M pro in a FRET-based activity assay. Comparatively, candesartan cilexetil had the highest selectivity index of all compounds tested as its half-maximal cytotoxicity concentration 50 (CC 50 ) value was the only one greater than the limit of the assay (>100 μM).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Autoregulation of the LIM kinases by their PDZ domain

LIM domain kinases (LIMK) are important regulators of actin cytoskeletal remodeling. These protein kinases phosphorylate the actin depolymerizing factor cofilin to suppress filament severing, and are key nodes between Rho GTPase cascades and actin. The two mammalian LIMKs, LIMK1 and LIMK2, contain consecutive LIM domains and a PDZ domain upstream of the C-terminal kinase domain. The roles of the N-terminal regions are not fully understood, and the function of the PDZ domain remains elusive. Here, we determine the 2.0 Å crystal structure of the PDZ domain of LIMK2 and reveal features not previously observed in PDZ domains including a core-facing arginine residue located at the second position of the ‘x-Φ-G-Φ’ motif, and that the expected peptide binding cleft is shallow and poorly conserved. We find a distal extended surface to be highly conserved, and when LIMK1 was ectopically expressed in yeast we find targeted mutagenesis of this surface decreases growth, implying increased LIMK activity. PDZ domain LIMK1 mutants expressed in yeast are hyperphosphorylated and show elevated activity in vitro. This surface in both LIMK1 and LIMK2 is critical for autoregulation independent of activation loop phosphorylation. Overall, our study demonstrates the functional importance of the PDZ domain to autoregulation of LIMKs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation

Paramyxoviruses are enveloped, nonsegmented, negative-strand RNA viruses that cause a wide spectrum of human and animal diseases. The viral genome, packaged by the nucleoprotein (N), serves as a template for the polymerase complex, composed of the large protein (L) and the homo-tetrameric phosphoprotein (P). The ~250-kDa L possesses all enzymatic activities necessary for its function but requires P in vivo. Structural information is available for individual P domains from different paramyxoviruses, but how P interacts with L and how that affects the activity of L is largely unknown due to the lack of high-resolution structures of this complex in this viral family. In this study we determined the structure of the L–P complex from parainfluenza virus 5 (PIV5) at 4.3-Å resolution using cryoelectron microscopy, as well as the oligomerization domain (OD) of P at 1.4-Å resolution using X-ray crystallography. P-OD associates with the RNA-dependent RNA polymerase domain of L and protrudes away from it, while the X domain of one chain of P is bound near the L nucleotide entry site. The methyltransferase (MTase) domain and the C-terminal domain (CTD) of L adopt a unique conformation, positioning the MTase active site immediately above the poly-ribonucleotidyltransferase domain and near the likely exit site for the product RNA 5' end. Our study reveals a potential mechanism that mononegavirus polymerases may employ to switch between transcription and genome replication. This knowledge will assist in the design and development of antivirals against paramyxoviruses.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The Q226L mutation can convert a highly pathogenic H5 2.3.4.4e virus to bind human-type receptors

H5Nx viruses continue to wreak havoc in avian and mammalian species worldwide. The virus distinguishes itself by the ability to replicate to high titers and transmit efficiently in a wide variety of hosts in diverse climatic environments. Fortunately, transmission to and between humans is scarce. Yet, if such an event were to occur, it could spark a pandemic as humans are immunologically naïve to H5 viruses. A significant determinant of transmission to and between humans is the ability of the influenza A virus hemagglutinin (HA) protein to shift from an avian-type to a human-type receptor specificity. Here, we demonstrate that a 2016 2.3.4.4e virus HA can convert to human-type receptor binding via a single Q226L mutation, in contrast to a cleavage-modified 2016 2.3.4.4b virus HA. Using glycan arrays, X-ray structural analyses, tissue- and direct glycan binding, we show that L133a Δ and 227Q are vital for this phenotype. Thus, whereas the 2.3.4.4e virus HA only needs a single amino acid mutation, the modified 2016 2.3.4.4b HA was not easily converted to human-type receptor specificity.

Science & Technology - Other Topics↗
DOE OSTI2020

Inhibition of DNA methylation in P. soloecismus alters algae productivity

Eukaryotic organisms regulate the organization, structure, and accessibility of their genomes through chromatin remodeling that can be inherited as epigenetic modifications. These DNA and histone protein modifications are ultimately responsible for an organism’s molecular adaptation to the environment, resulting in distinctive phenotypes. Epigenetic manipulation of algae holds yet untapped potential for the optimization of biofuel production and bioproduct formation; however, epigenetic machinery and modes-of-action have not been well characterized in algae. We sought to determine the extent to which the biofuel platform species Picochlorum soloecismus utilizes DNA methylation to regulate its genome. We found candidate genes with domains for DNA methylation in the P. soloecismus genome. Whole-genome bisulfite sequencing revealed DNA methylation in all three cytosine contexts (CpG, CHH, and CHG). While global DNA methylation is low overall (~1.15%), it occurs in appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution in all genomic contexts, though terminators contain the greatest number of CpG sites per kilobase. The P. soloecismus genome becomes hypomethylated during the growth cycle in response to nitrogen starvation. Algae cultures were treated daily across the growth cycle with 20 μM 5-aza-2'-deoxycytidine (5AZA) to inhibit propagation of DNA methylation in daughter cells. 5AZA treatment significantly increased optical density and forward and side scatter of cells across the growth cycle (16 days). This increase in cell size and complexity correlated with a significant increase (~66%) in lipid accumulation. Site specific CpG DNA methylation was significantly altered with 5AZA treatment over the time course, though nitrogen starvation itself induced significant hypomethylation in CpG contexts. Genes involved in several biological processes, including fatty acid synthesis, had altered methylation ratios in response to 5AZA; we hypothesize that these changes are potentially responsible for the phenotype of early induction of carbon storage as lipids. This is the first report to utilize epigenetic manipulation strategies to alter algal physiology and phenotype. Collectively, these data suggest these strategies can be utilized to fine-tune metabolic responses, alter growth, and enhance environmental adaption of microalgae for desired outcomes.

5-aza-2'-deoxycytidine↗
DOE OSTI2022

Structure of a dimeric photosystem II complex from a cyanobacterium acclimated to far-red light

Photosystem II (PSII) is the water-splitting enzyme central to oxygenic photosynthesis. To drive water oxidation, light is harvested by accessory pigments, mostly chlorophyll (Chl) a molecules, which absorb visible light (400–700 nm). Some cyanobacteria facultatively acclimate to shaded environments by altering their photosynthetic machinery to additionally absorb far-red light (FRL, 700–800 nm), a process termed far-red light photoacclimation or FaRLiP. During far-red light photoacclimation, FRL-PSII is assembled with FRL-specific isoforms of the subunits PsbA, PsbB, PsbC, PsbD, and PsbH, and some Chl-binding sites contain Chls d or f instead of the usual Chl a. The structure of an apo-FRL-PSII monomer lacking the FRL-specific PsbH subunit has previously been determined, but visualization of the dimeric complex has remained elusive. Here, we report the cryo-EM structure of a dimeric FRL–PSII complex. The site assignments for Chls d and f are consistent with those assigned in the previous apo-FRL-PSII monomeric structure. All sites that bind Chl d or Chl f at high occupancy exhibit a FRL-specific interaction of the formyl moiety of the Chl d or Chl f with the protein environment, which in some cases involves a phenylalanine sidechain. The structure retains the FRL-specific PsbH2 subunit, which appears to alter the energetic landscape of FRL-PSII, redirecting energy transfer from the phycobiliprotein complex to a Chl f molecule bound by PsbB2 that acts as a bridge for energy transfer to the electron transfer chain. Collectively, these observations extend our previous understanding of the structure-function relationship that allows PSII to function using lower energy FRL.

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

Visualization of the HIV-1 Env glycan shield across scales

The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the “glycan shield,” is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure–function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

CERES : a cryo-EM re-refinement system for continuous improvement of deposited models

The field of electron cryomicroscopy (cryo-EM) has advanced quickly in recent years as the result of numerous technological and methodological developments. This has led to an increase in the number of atomic structures determined using this method. Recently, several tools for the analysis of cryo-EM data and models have been developed within the Phenix software package, such as phenix.real_space_refine for the refinement of atomic models against real-space maps. Also, new validation metrics have been developed for low-resolution cryo-EM models. To understand the quality of deposited cryo-EM structures and how they might be improved, models deposited in the Protein Data Bank that have map resolutions of better than 5 Å were automatically re-refined using current versions of Phenix tools. The results are available on a publicly accessible web page (https://cci.lbl.gov/ceres). The implementation of a Cryo-EM Re-refinement System ( CERES ) for the improvement of models deposited in the wwPDB, and the results of the re-refinements, are described. Based on these results, contents are proposed for a `cryo-EM Table 1', which summarizes experimental details and validation metrics in a similar way to `Table 1' in crystallography. The consistent use of robust metrics for the evaluation of cryo-EM models and data should accompany every structure deposition and be reported in scientific publications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structural insights into protection against a SARS-CoV-2 spike variant by T cell receptor diversity

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell immunity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2 T cell epitopes elicit clonally restricted CD8 + T cell responses characterized by T cell receptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2–restricted spike protein epitope (RLQ) that elicits CD8 + T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recognizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ–HLA-A2 and T1006I–HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structural and Proteomic Studies of the Aureococcus anophagefferens Virus Demonstrate a Global Distribution of Virus-Encoded Carbohydrate Processing

Viruses modulate the function(s) of environmentally relevant microbial populations, yet considerations of the metabolic capabilities of individual virus particles themselves are rare. We used shotgun proteomics to quantitatively identify 43 virus-encoded proteins packaged within purified Aureococcus anophagefferens Virus (AaV) particles, normalizing data to the per-virion level using a 9.5-Å-resolution molecular reconstruction of the 1900-Å (AaV) particle that we generated with cryogenic electron microscopy. This packaged proteome was used to determine similarities and differences between members of different giant virus families. We noted that proteins involved in sugar degradation and binding (e.g., carbohydrate lyases) were unique to AaV among characterized giant viruses. To determine the extent to which this virally encoded metabolic capability was ecologically relevant, we examined the TARA Oceans dataset and identified genes and transcripts of viral origin. Our analyses demonstrated that putative giant virus carbohydrate lyases represented up to 17% of the marine pool for this function. In total, our observations suggest that the AaV particle has potential prepackaged metabolic capabilities and that these may be found in other giant viruses that are widespread and abundant in global oceans.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Facile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature

Abstract Although structures of vitrified supramolecular complexes have been determined at near-atomic resolution, elucidating in situ molecular structure in living cells remains a challenge. Here, we report a straightforward liquid cell technique, originally developed for real-time visualization of dynamics at a liquid-gas interface using transmission electron microscopy, to image wet biological samples. Due to the scattering effects from the liquid phase, the micrographs display an amplitude contrast comparable to that observed in negatively stained samples. We succeed in resolving subunits within the protein complex GroEL imaged in a buffer solution at room temperature. Additionally, we capture various stages of virus cell entry, a process for which only sparse structural data exists due to their transient nature. To scrutinize the morphological details further, we used individual particle electron tomography for 3D reconstruction of each virus. These findings showcase this approach potential as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Pixel-Registered Multimodal Synchrotron XRF and FTIR Microscopies Reveal Salinity Stress Response Mechanisms in Pistachio

Background: Salinity is a major abiotic stress that negatively affects nearly all plant species at all stages of growth. Drought and poor-quality irrigation cause high soil salinity and salt accumulation via evaporation, reducing crop productivity. Despite its critical importance, the spatial localization of salt ions and associated biochemical changes within plants experiencing high salinity remains largely unknown. In this study, we developed a multimodal imaging pipeline to understand the impact of salinity on the pistachio rootstock UCB-1 (Pistacia atlantica x Pistacia integerrima). We directly link biochemical fingerprints in stem tissue architecture with salt ion localization to provide insights into the strategies pistachio uses to tolerate salinity. Results: We observed that Pistacia spp. exposed to high salt conditions accumulated Ca, Si, Cl, Al and Mg as hotspots within the pith, compared to the control (of which only Ca and Al co-locate). In contrast, there was a decrease in K between the control and salinity treatment. Hotspots of amide I and II were present in the cortex and pith of the salinity treated sample. Additionally, the salinity treatment resulted in an increased abundance of pectin and carbohydrates within the pith compared to the control, and the abundance of esters/carboxylic acid was greater in the salinity treatment. Conclusions: We determined that Cl and K, S and P, and biochemical components polysaccharide and pectin, esters and carboxylic acid, amide I and cellulose are the strongest drivers of salinity- treatment induced variability. In the cortex and phloem/xylem, a negative K-Ca correlation decreases in the salinity treatment. Several hotspots of elements and amide I (proteins) appear under salinity treatment, particularly in the cortex, suggesting an increase in the production of stress-related proteins (in response to high Cl) and/or structural proteins (i.e. Ca). Together, these results indicate that pistachio responds to salinity through ion compartmentalization coupled with a targeted biochemical adjustment, rather than a broadscale tissue-wide response. Overall, these novel, spatially resolved pixel-registered multimodal imaging data provide an enabling platform to understand the mechanisms of salinity tolerance in Pistacia spp and can be broadly applied to studying stress-related phenotype response in various plant tissues.

FTIR spectromicroscopy↗
DOE OSTI2023

Structure-based mechanism and inhibition of cholesteryl ester transfer protein

Purpose of Review: Cholesteryl ester transfer proteins (CETP) regulate plasma cholesterol levels by transferring cholesteryl esters (CEs) among lipoproteins. Lipoprotein cholesterol levels correlate with the risk factors for atherosclerotic cardiovascular disease (ASCVD). This article reviews recent research on CETP structure, lipid transfer mechanism, and its inhibition. Recent Findings: Genetic deficiency in CETP is associated with a low plasma level of low-density lipoprotein cholesterol (LDL-C) and a profoundly elevated plasma level of high-density lipoprotein cholesterol (HDL-C), which correlates with a lower risk of atherosclerotic cardiovascular disease (ASCVD). However, a very high concentration of HDL-C also correlates with increased ASCVD mortality. Considering that the elevated CETP activity is a major determinant of the atherogenic dyslipidemia, i.e., pro-atherogenic reductions in HDL and LDL particle size, inhibition of CETP emerged as a promising pharmacological target during the past two decades. CETP inhibitors, including torcetrapib, dalcetrapib, evacetrapib, anacetrapib and obicetrapib, were designed and evaluated in phase III clinical trials for the treatment of ASCVD or dyslipidemia. Although these inhibitors increase in plasma HDL-C levels and/or reduce LDL-C levels, the poor efficacy against ASCVD ended interest in CETP as an anti-ASCVD target. Nevertheless, interest in CETP and the molecular mechanism by which it inhibits CE transfer among lipoproteins persisted. Insights into the structural-based CETP-lipoprotein interactions can unravel CETP inhibition machinery, which can hopefully guide the design of more effective CETP inhibitors that combat ASCVD. Summary: Individual-molecule 3D structures of CETP bound to lipoproteins provide a model for understanding the mechanism by which CETP mediates lipid transfer and which in turn, guide the rational design of new anti-ASCVD therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain

ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster . Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structures of methane and ammonia monooxygenases in native membranes

Methane- and ammonia-oxidizing bacteria play key roles in the global carbon and nitrogen cycles, respectively. These bacteria use homologous copper membrane monooxygenases to accomplish the defining chemical transformations of their metabolisms: the oxidations of methane to methanol by particulate methane monooxygenase (pMMO) and ammonia to hydroxylamine by ammonia monooxygenase (AMO), enzymes of prime interest for applications in mitigating climate change. However, investigations of these enzymes have been hindered by the need for disruptive detergent solubilization prior to structure determination, confounding studies of pMMO and precluding studies of AMO. Here, we overcome these challenges by using cryoEM to visualize pMMO and AMO directly in their native membrane arrays at 2.4 to 2.8 Å resolution. These structures reveal details of the copper centers, numerous bound lipids, and previously unobserved components, including identifiable and distinct supernumerary helices interacting with pMMO and AMO, suggesting a widespread role for these helices in copper membrane monooxygenases. Comparisons between these structures, their metallocofactors, and their unexpected protein–protein interactions highlight features that may govern activity or the formation of higher-order arrays in native membranes. The ability to obtain molecular insights within the native membrane will enable further understanding of these environmentally important enzymes.

Science & Technology - Other Topics↗