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At least 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5’ UTR of the viral genome

ABSTRACT Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for the formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the ubiquitin-like 1 (Ubl1), nucleic acid binding (NAB), β-coronavirus-specific marker (βSM) domains, and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5’ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5’ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5’ UTR of the viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein. IMPORTANCE The largest protein encoded by the SARS-CoV-2 genome is Nsp3. In infected cells, this multi-domain protein forms a pore structure in the virus-induced double-membrane vesicles (DMV). We have incomplete data on Nsp3 molecular structure, and here, we describe crystal structures for multiple domains of Nsp3. It is thought that newly replicated viral RNA transits through the DMV pore; however, we possess incomplete data on which regions of Nsp3 actually interact with RNA. Here, we present data showing that five domains of Nsp3 interact with the 5’ UTR of the SARS-CoV-2 RNA, including the Y domain for which no function has ever been discovered. These data suggest that the pore structure plays an active role in recognizing the terminal end of the genome, transiting and loading the viral RNA onto the cytoplasmic nucleocapsid protein. These data help expand our knowledge of Nsp3 structure and function and the SARS-CoV-2 replication cycle.

Microbiology↗
DOE OSTI2015

Simulation of Osmotic Swelling by the Stochastic Immersed Boundary Method

We develop here computational methods for the simulation of osmotic swelling phenomena relevant to microscopic vesicles containing transformable solute molecules. We introduce stochastic immersed boundary methods (SIBMs) that can capture osmotically driven fluid transport through semipermeable elastic membranes subject to thermal fluctuations. We further develop numerical methods to handle within SIBMs an elastic shell model for a neo-Hookean material. Our extended SIBM allows for capturing osmotic swelling phenomena driven by concentration changes and interactions between a discrete collection of confined particles while accounting for the thermal fluctuations of the semipermeable membrane and the hydrodynamic transport of solvent. We use our computational methods to investigate osmotic phenomena in regimes that go beyond the classical Van't Hoff theory. We develop statistical mechanics theories for osmotic swelling of vesicles when there are significant interactions between particles that can transform over time. We validate our theoretical results against detailed computational simulations. Our methods are expected to be useful for a wide class of applications allowing for the simulation of osmotically driven flows, thermally fluctuating semipermeable elastic structures, and solute interactions.

97 MATHEMATICS AND COMPUTING↗
DOE OSTI2016

Calorimetry-Derived Composition Vectors to Resolve Component Raman Spectra in Phospholipid Phase Transitions

Multidimensional least squares analysis is a well-established technique for resolving component vibrational spectra from mixed samples or systems. Component resolution of temperature-dependent vibrational spectra is challenging, however, due to the lack of a suitable model for the variation in sample composition with temperature. In this work, analysis of temperature-dependent Raman spectra of lipid membranes is accomplished by using “concentration” vectors independently derived from enthalpy changes determined by differential scanning calorimetry. Specifically, the lipid–bilayer phase transitions of DMPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) are investigated through Raman spectra acquired from individual, optically trapped vesicles in suspension as a function of temperature. Heat capacity profiles of the same vesicle suspension are measured using differential scanning calorimetry and numerically integrated to generate enthalpy change curves of each phase transition, which are in turn used to construct composition vectors. Multidimensional least squares analysis optimized for a fit to these composition vectors allows resolution of the component spectra corresponding to gel, ripple, and liquid–crystalline phases of the DMPC. The quality of fit of the calorimetry-derived results is confirmed by unstructured residual differences between the data and the model, and a composition variation predicted by the resolved spectra that matches the calorimetry results. Furthermore, this approach to analysis of temperature-dependent spectral data could be readily applied in other areas of materials characterization, where one is seeking to learn about structural changes that occur through temperature-dependent phase transitions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2014

Impaired osteoblast differentiation in Annexin A2- and -A5-deficient cells

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts inflammation,fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We generated annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and asked whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to vector controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and staining activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to controls. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. Using a murine fracture model, we demonstrate that AnxA2 and AnxA5 are rapidly expressed within the fracture callus. These data demonstrate that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation and differentiation in addition to their well-studied function in matrix vesicles.

annexin, bone, gene expression↗
DOE OSTI2014

Membrane-Organized Chemical Photoredox Systems

This project has three interrelated goals relevant to solar water photolysis, which are to develop: (1) vesicle-organized assemblies for H 2 photoproduction that utilize pyrylium and structurally related compounds as combined photosensitizers and cyclic electroneutral transmembrane electron carriers; (2) transmembrane redox systems whose reaction rates can be modulated by light; and (3) homogeneous catalysts for water oxidation. In area (1), initial efforts to photogenerate H 2 from vectorially-organized vesicles containing occluded colloidal Pt and commonly available pyrylium ions as transmembrane redox mediators were unsuccessful. New pyrylium compounds with significantly lower reduction potentials have been synthesized to address this problem, their apparent redox potentials in functioning systems have been now evaluated by using a series of occluded viologens, and H 2 photoproduction has been demonstrated in continuous illumination experiments. In area (2), spirooxazine-quinone dyads have been synthesized and their capacity to function as redox mediators across bilayer membranes has been evaluated through continuous photolysis and transient spectrophotometric measurements. Photoisomerization of the spiro moiety to the ring-open mero form caused net quantum yields to decrease significantly, providing a basis for photoregulation of transmembrane redox. Research on water oxidation (area 3) has been directed at understanding mechanisms of catalysis by cis,cis-[(bpy) 2 Ru(OH 2 )] 2 O 4+ and related polyimine complexes. Using a variety of physical techniques, we have: (i) identified the redox state of the complex ion that is catalytically active; (ii) shown using 18 O isotopic labeling that there are two reaction pathways, both of which involve participation of solvent H 2 O; and (iii) detected and characterized by EPR and resonance Raman spectroscopies new species which may be key intermediates in the catalytic cycle.

14 SOLAR ENERGY↗
DOE OSTI2022

Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein–protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Two VHH Antibodies Neutralize Botulinum Neurotoxin E1 by Blocking Its Membrane Translocation in Host Cells

Botulinum neurotoxin serotype E (BoNT/E) is one of the major causes of human botulism, which is a life-threatening disease caused by flaccid paralysis of muscles. After receptor-mediated toxin internalization into motor neurons, the translocation domain (HN) of BoNT/E transforms into a protein channel upon vesicle acidification in endosomes and delivers its protease domain (LC) across membrane to enter the neuronal cytosol. It is believed that the rapid onset of BoNT/E intoxication compared to other BoNT serotypes is related to its swift internalization and translocation. We recently identified two neutralizing single-domain camelid antibodies (VHHs) against BoNT/E1 termed JLE-E5 and JLE-E9. Here, we report the crystal structures of these two VHHs bound to the LCHN domain of BoNT/E1. The structures reveal that these VHHs recognize two distinct epitopes that are partially overlapping with the putative transmembrane regions on HN, and therefore could physically block membrane association of BoNT/E1. This is confirmed by our in vitro studies, which show that these VHHs inhibit the structural change of BoNT/E1 at acidic pH and interfere with BoNT/E1 association with lipid vesicles. Therefore, these two VHHs neutralize BoNT/E1 by preventing the transmembrane delivery of LC. Furthermore, structure-based sequence analyses show that the 3-dimensional epitopes of these two VHHs are largely conserved across many BoNT/E subtypes, suggesting a broad-spectrum protection against the BoNT/E family. In summary, this work improves our understanding of the membrane translocation mechanism of BoNT/E and paves the way for developing VHHs as diagnostics or therapeutics for the treatment of BoNT/E intoxication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Crystal structure of the receptor-binding domain of botulinum neurotoxin type HA, also known as type FA or H

Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Altogether, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Mechanistic insights into neurotransmitter release and presynaptic plasticity from the crystal structure of Munc13-1 C1C2BMUN

Munc13–1 acts as a master regulator of neurotransmitter release, mediating docking-priming of synaptic vesicles and diverse presynaptic plasticity processes. It is unclear how the functions of the multiple domains of Munc13–1 are coordinated. The crystal structure of a Munc13–1 fragment including its C1, C2B and MUN domains (C1C2BMUN) reveals a 19.5 nm-long multi-helical structure with the C1 and C2B domains packed at one end. The similar orientations of the respective diacyglycerol- and Ca2+-binding sites of the C1 and C2B domains suggest that the two domains cooperate in plasma-membrane binding and that activation of Munc13–1 by Ca2+ and diacylglycerol during short-term presynaptic plasticity are closely interrelated. Electrophysiological experiments in mouse neurons support the functional importance of the domain interfaces observed in C1C2BMUN. The structure imposes key constraints for models of neurotransmitter release and suggests that Munc13–1 bridges the vesicle and plasma membranes from the periphery of the membrane-membrane interface.

Xu, Junjie↗
NASA NTRS1975

Some studies of an unusual eucrite - Ibitira

The Ibitira eucrite is remarkable both for its vesicles and its unbrecciated nature. It consists of nearly 63 vol.% pyroxene (Wo14En38Fs48), 31% plagioclase (An95-96), nearly 2% of nickel-iron, troilite, ilmenite, titanian chromite, and 4% of a silica polymorph. It has a mean track density of 1.8 plus or minus 0.3 x 1,000,000 per sq cm, mainly due to cosmic rays. Its pre-atmospheric radius must have been at least 10 cm. The absence of complex radiation effects and presence of vesicles place constraints on the thickness of the Ibitira basalt flow. From the freezing time calculations of Provost and Bottinga, it appears that Ibitira came from a flow no less than 2.5 m and probably no more than 10 or 20 m thick. However, this estimate depends strongly on the viscosity of the melt, which is not well known.

Wilkening, L. L.↗
NASA NTRS1977

Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium halobium

Halobacterium halobium is known to contain sheets of bacteriorhodopsin, a pigment which upon exposure to light undergoes cyclic protonation and deprotonation, resulting in net H(+) translocation. In this paper, experiments were conducted to test H. halobium cell envelope vesicles for respiration-induced glutamate uptake. It is shown that glutamate transport in H. halobium cell envelope vesicles can occur as a result of respiration, as well as light acting on bacteriorhodopsin. Glutamate transport can be energized by the oxidation of dimethyl phenylenediamine, and the properties of the transport system are entirely analogous to those observed with illumination as the source of energy. In the case of respiration-dependent glutamate transport, the transportation is also driven by a Na(+) gradient, thereby confirming the existence of a single glutamate transport system independent of the source of energy. The analogy observed is indirect evidence that the cytochrome oxidase of H. halobium functions as a H(+) pump.

Belliveau, J. W.↗
NASA NTRS1976

Fluorine as a constituent in lunar magmatic gases

Fluorine surface deposits on Apollo 15 green glass, Apollo 17 orange glass, and on vesicle linings from Apollo 15 basalts were studied by a resonant nuclear reaction technique. Surface layers of about 10 to the 15th F atoms/sq cm were observed on unbroken spheroidal glass surfaces, whereas thinner (about 10 to the 14th atoms/sq cm) deposits were found on the vesicle linings.

Goldberg, R. H.↗
NASA NTRS1977

Experimental vaporization of the Holbrook chondrite

The vapor phase composition obtained by heating samples of the Holbrook L6 chondrite to 1300 C was determined quantitatively by Knudsen cell-quadrupole mass spectrometry. Maximum observed vapor pressures, produced at 1200 C, are reported for Na, K, Fe, and Ni, and the implications of the Na/K ratio are considered. The Fe and Ni data are discussed with attention to their migration in individual equilibrated chondrites. S2 (with minor SO2), H2O, and CO2 were also present in the high-temperature gas phase. Vesicles formed by the release of intrinsically derived volatiles are compared with vesicles in the Ibitira eucrite. Chondrite evolution is briefly discussed.

Gooding, J. L.↗
NASA NTRS1979

Gating effects in Halobacterium halobium membrane transport

The transport of Na(+) via an H(+)/Na(+) antiporter and of aspartate and serine via Na(+)/amino acid symport systems was studied in Halobacterium halobium cell envelope vesicles. Gradients for H(+) were produced by illuminating the bacteriorhodopsin-containing vesicles at different light intensities, and the rate and extent of Na(+) transport were followed as functions of the electrochemical potential difference for protons. The coupling of Na(+) and H(+) gradients suggested a translocation stoichiometry of 2H(+)/Na(+) for the antiporter. The rate of Na(+) transport increases steeply above a critical transmembrane electrochemical proton gradient, and since the electrical and the chemical potentials of H(+) at this threshold point vary with the experimental conditions, while the sum of these potentials is constant, it was concluded that the gating of the Na(+) transport is caused by the total electrochemical gradient.

Lanyi, J. K.↗
NASA NTRS1979

Light-driven solute transport in Halobacterium halobium

The cell membrane of Halobacterium halobium exhibits differential regions which contain crystalline arrays of a single kind of protein, termed bacteriorhodopsin. This bacterial retinal-protein complex resembles the visual pigment and, after the absorption of protons, translocates H(+) across the cell membrane, leading to an electrochemical gradient for protons between the inside and the outside of the cell. Thus, light is an alternate source of energy in these bacteria, in addition to terminal oxidation. The paper deals with work on light-driven transport in H. halobium with cell envelope vesicles. The discussion covers light-driven movements of H(+), Na(+), and K(+); light-driven amino acid transport; and apparent allosteric control of amino acid transport. The scheme of energy coupling in H. halobium vesicles appears simple, its quantitative details are quite complex and reveal regulatory phenomena. More knowledge is required of the way the coupling components are regulated by the ion gradients present.

Lanyi, J. K.↗
NASA NTRS1982

Magma vesiculation and pyroclastic volcanism on Venus

Theoretical consideration of the magma vesiculation process under observed and inferred venusian surface conditions suggests that vesicles should form in basaltic melts, especially if CO2 is the primary magmatic volatile. However, the high surface atmospheric pressure (about 90 bars) and density on Venus retard bubble coalescence and disruption sufficiently to make explosive volcanism unlikely. The products of explosive volcanism (fire fountains, convecting eruption clouds, pyroclastic flows, and topography-mantling deposits of ash, spatter, and scoria) should be rare on Venus, and effusive eruptions should dominate. The volume fraction of vesicles in basaltic rocks on Venus are predicted to be less than in chemically similar rocks on earth. Detection of pyroclastic landforms or eruption products on Venus would indicate either abnormally high volatile contents of Venus magmas (2.5-4 wt%) or different environmental conditions (e.g., lower atmospheric pressure) in previous geologic history.

Garvin, J. B.↗
NASA NTRS1982

Effect of disuse on sarcoplasmic reticulum in fast and slow skeletal muscle

The effect of 6 weeks of hindlimb immobilization on rat skeletal muscle sarcoplasmic reticulum (SR) was determined in the slow-twitch, type 1 soleus (SOL), the fast-twitch, type 2A deep region of the vastus lateralis (DVL), and the fast-twitch, type 2B superficial region of the vastus lateralis (SVL). Immobilization produced a significant decline in the Ca(2+) uptake rate (V sub max) of SR vesicles from the slow SOL, while the SR V sub max increased in the fast SVL and was unaltered in the DVL. Vesicles from the fast SVL and DVL also exhibited a higher total Ca(2+) uptake capacity following immobilization. An evaluation of the time course of the immobilization-mediated effect revealed an increased Ca(2+) uptake capacity in all three samples after 1 wk. In the SOL total Ca(2+) uptake returned to control level after 2 wk, while in the fast-twitch muscles the higher capacities were maintained. The Ca(2+)-stimulated SR ATPase activity was not altered in any of the muscle studies.

Kim, D. H.↗
NASA NTRS1984

Mineralogical comparison and cooling history of lunar and chondritic vesicular melt breccias

Lunar sample 77135, an impact melt breccia full of vesicles, has been reinvestigated by electron microprobe and X-ray diffraction techniques and compared with a vesicular melt LL chondrite, Yamato 790964, in an attempt to understand their impact heating processes and subsequent cooling history. Notable similarities between the lunar and chondritic melt breccias include: abundant vesicles, similar pyroxene chemical zoning trends, the presence of variable amounts of clastic material, and similar chemical compositions except for K and Na contents of glass and mesostasis. Some constraints on the cooling history are estimated from Mg-Fe diffusion profiles in olivine and pyroxene. The burial depth of lunar sample 77135 during cooling was 0.2-100 m; the depth for the chondrite was probably smaller. Impact melts were probably produced and a layer of regolith retained on the parent body sufficiently thick to allow the olivines to homogenize during slow cooling.

Miyamoto, M.↗