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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 343 records · Page 19
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

VAMPs sensitive to tetanus toxin are required for cortical granule exocytosis in mouse oocytes

Highlights: • Cortical granule exocytosis is sensitive to tetanus toxin in mouse oocytes. • VAMP1 and VAMP3 proteins participate in cortical granule exocytosis. • This work confirms that cortical granule exocytosis is mediated by SNARE complex. Fusion of cortical granules with oocyte plasma membrane is one of the most significant secretory events to prevent polyspermy during oocyte activation. Cortical granule exocytosis (CGE) is distinct from most other exocytosis because cortical granules are not renewed after secretion. However, it is thought to be mediated by SNARE complex, which mediates membrane fusion in other exocytoses. SNAREs proteins are divided into Q (glutamine)- and R (arginine)-SNAREs. Q-SNAREs include Syntaxins and SNAP25 family, and R-SNAREs include VAMPs family. In mouse oocytes, Syntaxin4 and SNAP23 have been involved in CGE; nevertheless, it is unknown if VAMP is required. Here, we demonstrated by RT-PCR and immunoblotting that VAMP1 and VAMP3 are expressed in mouse oocyte, and they localized in the cortical region of this cell. Using a functional assay to quantify CGE, we showed that tetanus toxin –which specifically cleavages VAMP1, VAMP2 or VAMP3– inhibited CGE suggesting that at least one VAMP was necessary. Function blocking assays demonstrated that only the microinjection of anti-VAMP1 or anti-VAMP3 antibodies abolished CGE in activated oocytes. These findings demonstrate that R-SNAREs sensitive to tetanus toxin, VAMP1 and VAMP3 –but not VAMP2-, are required for CGE and demonstrate that CGE is mediated by the SNARE complex.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

De novo design of monomeric helical bundles for pH ‐controlled membrane lysis

Abstract Targeted intracellular delivery via receptor‐mediated endocytosis requires the delivered cargo to escape the endosome to prevent lysosomal degradation. This can in principle be achieved by membrane lysis tightly restricted to endosomal membranes upon internalization to avoid general membrane insertion and lysis. Here, we describe the design of small monomeric proteins with buried histidine containing pH‐responsive hydrogen bond networks and membrane permeating amphipathic helices. Of the 30 designs that were experimentally tested, all expressed in Escherichia coli , 13 were monomeric with the expected secondary structure, and 4 designs disrupted artificial liposomes in a pH‐dependent manner. Mutational analysis showed that the buried histidine hydrogen bond networks mediate pH‐responsiveness and control lysis of model membranes within a very narrow range of pH (6.0–5.5) with almost no lysis occurring at neutral pH. These tightly controlled lytic monomers could help mediate endosomal escape in designed targeted delivery platforms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Functional lipid pairs as building blocks of phase-separated membranes

Biological membranes exhibit a great deal of compositional and phase heterogeneity due to hundreds of chemically distinct components. As a result, phase separation processes in cell membranes are extremely difficult to study, especially at the molecular level. It is currently believed that the lateral membrane heterogeneity and the formation of domains, or rafts, are driven by lipid–lipid and lipid–protein interactions. Nevertheless, the underlying mechanisms regulating membrane heterogeneity remain poorly understood. In the present work, we combine inelastic X-ray scattering with molecular dynamics simulations to provide direct evidence for the existence of strongly coupled transient lipid pairs. These lipid pairs manifest themselves experimentally through optical vibrational (a.k.a. phononic) modes observed in binary (1,2-dipalmitoyl- sn -glycero-3-phosphocholine [DPPC]–cholesterol) and ternary (DPPC–1,2-dioleoyl- sn -glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine [DOPC/POPC]–cholesterol) systems. The existence of a phononic gap in these vibrational modes is a direct result of the finite size of patches formed by these lipid pairs. The observation of lipid pairs provides a spatial (subnanometer) and temporal (subnanosecond) window into the lipid–lipid interactions in complex mixtures of saturated/unsaturated lipids and cholesterol. Our findings represent a step toward understanding the lateral organization and dynamics of membrane domains using a well-validated probe with a high spatial and temporal resolution.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Reagent to label proteins via lysine isopeptide bonds

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction—first, cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate, and second, joining the terminal threonine to the nucleophilic lysine residue residing within the pilin motif of another pilin protomer. Informed by the high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and by developing structural variants of the sortase enzyme whose catalytic pocket has been unmasked by activating mutations, we have developed new reagents capable of forming isopeptide bonds in vitro. The reagents disclosed herein can catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile new platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Clubb, Robert T.↗
DOE OSTI2020

Water Structure and Properties at Hydrophilic and Hydrophobic Surfaces

The properties of water on both molecular and macroscopic surfaces critically influence a wide range of physical behaviors, with applications spanning from membrane science to catalysis to protein engineering. Yet, our current understanding of water interfacing molecular and material surfaces is incomplete, in part because measurement of water structure and molecular-scale properties challenges even the most advanced experimental characterization techniques and computational approaches. This review highlights progress in the ongoing development of tools working to answer fundamental questions on the principles that govern the interactions between water and surfaces. One outstanding and critical question is what universal molecular signatures capture the hydrophobicity of different surfaces in an operationally meaningful way, since traditional macroscopic hydrophobicity measures like contact angles fail to capture even basic properties of molecular or extended surfaces with any heterogeneity at the nanometer length scale. Resolving this grand challenge will require close interactions between state-of-the-art experiments, simulations, and theory, spanning research groups and using agreed-upon model systems, to synthesize an integrated knowledge of solvation water structure, dynamics, and thermodynamics.

catalysis (heterogeneous)↗
DOE OSTI2026

LHCSR1 Functions as a Dimmer Switch for Light Harvesting

In oxygenic photosynthesis, high light leads to a set of photoprotective processes, known as nonphotochemical quenching, that are required for fitness. In moss and algae, the pigment−protein complex, light-harvesting stress-related (LHCSR), is crucial for photoprotection. Acidification of the thylakoid lumen under high light triggers the activation of LHCSR and the conversion of the xanthophyll violaxanthin into zeaxanthin, which is found within LHCSR. These interrelated molecular components combine to turn on a safety valve that dissipates excess energy as heat. Previous studies of detergent-solubilized LHCSR1 found that pH and zeaxanthin regulate distinct quenching sites via the protein conformation. However, protein function in the native membrane environment can be significantly different. In this work, we applied single-molecule fluorescence spectroscopy to LHCSR1 in membrane nanodiscs. The membrane environment enhanced total quenching, regardless of pH or zeaxanthin-binding. pH-dependent quenching was still observed whereas zeaxanthin-dependent quenching was suppressed. Conformational changes also increased in the membrane, establishing that the local environment can change the nature of the equilibrium between light harvesting and photoprotection.

Hoffmann, Madeline P. [Massachusetts Inst. of Tech↗
DOE OSTI2020

Structure of the respiratory MBS complex reveals iron-sulfur cluster catalyzed sulfane sulfur reduction in ancient life

Abstract Modern day aerobic respiration in mitochondria involving complex I converts redox energy into chemical energy and likely evolved from a simple anaerobic system now represented by hydrogen gas-evolving hydrogenase (MBH) where protons are the terminal electron acceptor. Here we present the cryo-EM structure of an early ancestor in the evolution of complex I, the elemental sulfur (S 0 )-reducing reductase MBS. Three highly conserved protein loops linking cytoplasmic and membrane domains enable scalable energy conversion in all three complexes. MBS contains two proton pumps compared to one in MBH and likely conserves twice the energy. The structure also reveals evolutionary adaptations of MBH that enabled S 0 reduction by MBS catalyzed by a site-differentiated iron-sulfur cluster without participation of protons or amino acid residues. This is the simplest mechanism proposed for reduction of inorganic or organic disulfides. It is of fundamental significance in the iron and sulfur-rich volcanic environments of early earth and possibly the origin of life. MBS provides a new perspective on the evolution of modern-day respiratory complexes and of catalysis by biological iron-sulfur clusters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural insights into the disruption of TNF-TNFR1 signalling by small molecules stabilising a distorted TNF

Tumour necrosis factor (TNF) is a trimeric protein which signals through two membrane receptors, TNFR1 and TNFR2. Previously, we identified small molecules that inhibit human TNF by stabilising a distorted trimer and reduce the number of receptors bound to TNF from three to two. Here we present a biochemical and structural characterisation of the small molecule-stabilised TNF-TNFR1 complex, providing insights into how a distorted TNF trimer can alter signalling function. We demonstrate that the inhibitors reduce the binding affinity of TNF to the third TNFR1 molecule. In support of this, we show by X-ray crystallography that the inhibitor-bound, distorted, TNF trimer forms a complex with a dimer of TNFR1 molecules. This observation, along with data from a solution-based network assembly assay, leads us to suggest a model for TNF signalling based on TNF-TNFR1 clusters, which are disrupted by small molecule inhibitors.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

The linker protein ApcI regulates light harvesting under red light in Synechocystis sp. PCC 6803

Phycobilisomes (PBSs) are versatile cyanobacterial antenna complexes that harvest light energy to drive photosynthesis. They can adapt to various light conditions; for example, dismantling under high light to prevent photo-oxidation and arranging in rows under low light to increase light harvesting efficiency. Light quality also influences PBS structure and function, as observed under far-red light exposure. Here, we describe a PBS linker protein, ApcI (previously hypothetical protein Sll1911), expressed specifically under red light (620 nm) or upon chemically induced reduction of the plastoquinone pool. We characterized ApcI in Synechocystis sp. PCC 6803 using mutant analyses, PBS binding experiments, and protein interaction studies. Deletion of apcI conferred high light tolerance on Synechocystis sp. PCC 6803 compared to the wild-type strain, leading to reduced energy transfer from PBSs to the photosystems under high light. Binding experiments revealed that ApcI replaces the linker protein ApcG at the membrane-facing side of the PBS core via a paralogous C-terminal motif. Additionally, the N-terminal region of ApcI interacts with photosystem II. Our findings highlight the importance of PBS remodeling for adaptation to different light conditions. The characterization of ApcI provides insight into the mechanisms by which cyanobacteria optimize light harvesting in response to varying light conditions.

Photosystem↗
DOE OSTI2021

A Novel Mode of Photoprotection Mediated by a Cysteine Residue in the Chlorophyll Protein IsiA

Oxygenic photosynthetic organisms have evolved a multitude of mechanisms for protection against high-light stress. IsiA, a chlorophyll a-binding cyanobacterial protein, serves as an accessory antenna complex for photosystem I. Intriguingly, IsiA can also function as an independent pigment protein complex in the thylakoid membrane and facilitate the dissipation of excess energy, providing photoprotection. The molecular basis of the IsiA-mediated excitation quenching mechanism remains poorly understood. In this study, we demonstrate that IsiA uses a novel cysteine-mediated process to quench excitation energy. The single cysteine in IsiA in the cyanobacterium Synechocystis sp. strain PCC 6803 was converted to a valine. Ultrafast fluorescence spectroscopic analysis showed that this single change abolishes the excitation energy quenching ability of IsiA, thus providing direct evidence of the crucial role of this cysteine residue in energy dissipation from excited chlorophylls. Under stress conditions, the mutant cells exhibited enhanced light sensitivity, indicating that the cysteine-mediated quenching process is critically important for photoprotection.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Proteomic Insights into Trichome Responses to Elevated Elemental Stress in Cation Exchanger (CAX) Mutants

Abstract Research on elemental distribution in plants is crucial for understanding nutrient uptake, environmental adaptation and optimizing agricultural practices for sustainable food production. Plant trichomes, with their self-contained structures and easy accessibility, offer a robust model system for investigating elemental repartitioning. Transport proteins, such as the four functional cation exchangers (CAXs) in Arabidopsis, are low-affinity, high-capacity transporters primarily located on the vacuole. Mutants in these transporters have been partially characterized, one of the phenotypes of the CAX1 mutant being altered with tolerance to low-oxygen conditions. A simple visual screen demonstrated trichome density and morphology in cax1, and quadruple CAX (cax1-4: qKO) mutants remained unaltered. Here, we used synchrotron X-ray fluorescence (SXRF) to show that trichomes in CAX-deficient lines accumulated high levels of chlorine, potassium, calcium and manganese. Proteomic analysis on isolated Arabidopsis trichomes showed changes in protein abundance in response to changes in element accumulation. The CAX mutants showed an increased abundance of plasma membrane ATPase and vacuolar H-pumping proteins, and proteins associated with water movement and endocytosis, while also showing changes in proteins associated with the regulation of plasmodesmata. These findings advance our understanding of the integration of CAX transport with elemental homeostasis within trichomes and shed light on how plants modulate protein abundance under conditions of altered elemental levels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Non-destructive spatial analysis of phosphatase activity and total protein distribution in the rhizosphere using a root blotting method

Phosphorus (P) is an essential macronutrient for plant growth, but bioavailable P in soils is often limited due to immobilization resulting from pH and geochemical interactions. Understanding the dynamics of P in soils and elucidating the mechanisms by which plants access P from their environment are critical to evaluating productivity, particularly in nutrient poor environments. Phosphorus from organic matter can act as a major source of P for organisms in soil systems. Phosphatases, enzymes that liberate inorganic P from organic sources, are produced by both plants and microbes and are considered one of the most active classes of enzymes in soil. In this work we developed a root blotting method to spatially image phosphatase activity in the rhizosphere. Proteins from the rhizosphere are transferred to a nitrocellulose membrane while retaining their enzymatic activity and two-dimensional spatial distribution. Subsequent application of a fluorogenic phosphatase indicator, DDAO phosphate, enables visualization of the distribution of phosphatase activity in the sample. The proteins can then be fixed to the membrane and treated with a SYPRO® Ruby gel stain, a fluorescent total protein stain, allowing for visualization of total protein distribution. Taken together, the images of phosphatase activity and total protein localization can be mapped back to the root architecture and provide insight into factors affecting the spatial distribution of enzymatic activity and protein accumulation in the rhizosphere. Notably, this method can be applied to plants growing in rhizoboxes containing soil or soilless growth mixtures (e.g., sand or various potting mixes) and, because of the non-destructive nature of this approach, be performed over time to track changes. We anticipate that this fluorescent indicator imaging technique on root blots can be used in diverse plant-microbe-soil systems to better understand the role of phosphatases in P acquisition and soil P cycling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Brominated lipid probes expose structural asymmetries in constricted membranes

Lipids in biological membranes are thought to be functionally organized, but few experimental tools can probe nanoscale membrane structure. Using brominated lipids as contrast probes for cryo-EM and a model ESCRT-III membrane-remodeling system composed of human CHMP1B and IST1, we observed leaflet-level and protein-localized structural lipid patterns within highly constricted and thinned membrane nanotubes. These nanotubes differed markedly from protein-free, flat bilayers in leaflet thickness, lipid diffusion rates and lipid compositional and conformational asymmetries. Simulations and cryo-EM imaging of brominated stearoyl-docosahexanenoyl-phosphocholine showed how a pair of phenylalanine residues scored the outer leaflet with a helical hydrophobic defect where polyunsaturated docosahexaenoyl tails accumulated at the bilayer surface. Combining cryo-EM of halogenated lipids with molecular dynamics thus enables new characterizations of the composition and structure of membranes on molecular length scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Overexpression of human ACE2 protein in mouse fibroblasts stably transfected with the intact ACE2 gene

Infection by SARS-CoV-2 is dependent on binding of the viral spike protein to angiotensin converting enzyme 2 (ACE2), a membrane glycoprotein expressed on epithelial cells in the human upper respiratory tract. Recombinant ACE2 protein has potential application for anti-viral therapy. Here we co-transfected mouse fibroblasts (A9 cells) with a cloned fragment of human genomic DNA containing the intact ACE2 gene and an unlinked neomycin phosphotransferase gene, and then selected stable neomycin-resistant transfectants. Transfectant clones expressed ACE2 protein at levels that were generally proportional to the number of ACE2 gene copies integrated in the cell genome, ranging up to approximately 50 times the level of ACE2 present of Vero-E6 cells. Cells overexpressing ACE2 were hypersensitive to infection by spike-pseudotyped vesicular stomatitis virus (VSV-S), and adsorption of VSV-S to these cells occurred at an accelerated rate compared to Vero-E6 cells. In conclusion, the transfectant cell clones described here therefore have favorable attributes as feedstocks for large-scale production of recombinant human ACE2 protein.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2020

Phosphatidic acid: an emerging versatile class of cellular mediators

Abstract Lipids function not only as the major structural components of cell membranes, but also as molecular messengers that transduce signals to trigger downstream signaling events in the cell. Phosphatidic acid (PA), the simplest and a minor class of glycerophospholipids, is a key intermediate for the synthesis of membrane and storage lipids, and also plays important roles in mediating diverse cellular and physiological processes in eukaryotes ranging from microbes to mammals and higher plants. PA comprises different molecular species that can act differently, and is found in virtually all organisms, tissues, and organellar membranes, with variations in total content and molecular species composition. The cellular levels of PA are highly dynamic in response to stimuli and multiple enzymatic reactions can mediate its production and degradation. Moreover, its unique physicochemical properties compared with other glycerophospholipids allow PA to influence membrane structure and dynamics, and interact with various proteins. PA has emerged as a class of new lipid mediators modulating various signaling and cellular processes via its versatile effects, such as membrane tethering, conformational changes, and enzymatic activities of target proteins, and vesicular trafficking.

Biochemistry & Molecular Biology↗
DOE OSTI2020

Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ~90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.

KRAS↗
DOE OSTI2023

Physiological potential and evolutionary trajectories of syntrophic sulfate-reducing bacterial partners of anaerobic methanotrophic archaea

Sulfate-coupled anaerobic oxidation of methane (AOM) is performed by multicellular consortia of anaerobic methanotrophic archaea (ANME) in obligate syntrophic partnership with sulfate-reducing bacteria (SRB). Diverse ANME and SRB clades co-associate but the physiological basis for their adaptation and diversification is not well understood. In this work, we used comparative metagenomics and phylogenetics to investigate the metabolic adaptation among the 4 main syntrophic SRB clades (HotSeep-1, Seep-SRB2, Seep-SRB1a, and Seep-SRB1g) and identified features associated with their syntrophic lifestyle that distinguish them from their non-syntrophic evolutionary neighbors in the phylum Desulfobacterota. We show that the protein complexes involved in direct interspecies electron transfer (DIET) from ANME to the SRB outer membrane are conserved between the syntrophic lineages. In contrast, the proteins involved in electron transfer within the SRB inner membrane differ between clades, indicative of convergent evolution in the adaptation to a syntrophic lifestyle. Our analysis suggests that in most cases, this adaptation likely occurred after the acquisition of the DIET complexes in an ancestral clade and involve horizontal gene transfers within pathways for electron transfer (CbcBA) and biofilm formation (Pel). We also provide evidence for unique adaptations within syntrophic SRB clades, which vary depending on the archaeal partner. Among the most widespread syntrophic SRB, Seep-SRB1a, subclades that specifically partner ANME-2a are missing the cobalamin synthesis pathway, suggestive of nutritional dependency on its partner, while closely related Seep-SRB1a partners of ANME-2c lack nutritional auxotrophies. Our work provides insight into the features associated with DIET-based syntrophy and the adaptation of SRB towards it.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

In Vitro Selection of Antibodies Targeting Yersinia pestis Membrane Lipids Using Nanodisc-Based Antigen Presentation

Proteins are the most common targets for antibody discovery and vaccine development, but their sequence variability can limit the breadth of resulting antigens. Lipids represent an alternative class of antigens due to their structural conservation and roles in host–pathogen interactions. Here, we describe the development and optimization of an in vitro antibody selection workflow using lipid-containing nanodiscs as antigen presentation platforms to enable phage and yeast display selections under conditions adapted for these non-protein targets. Lipopolysaccharide (LPS) nanodiscs were first used as a model system to evaluate selection strategies, including competitive and subtractive approaches to reduce non-specific binders, yielding peptide and single-chain variable fragment (scFv) binders that were affinity matured to improve binding signals. The same approach was subsequently used to select scFv antibodies that recognize lipid nanodiscs prepared from Yersinia pestis membrane lipid extracts. These antibodies show binding to lipid nanodiscs derived from Y. pestis, with evidence of selectivity relative to control nanodiscs. Overall, this work establishes a workflow for antibody selection against lipid-containing nanodisc antigens and highlights practical considerations associated with these targets. The approach may be useful for generating affinity reagents to membrane-associated lipids, although further characterization is required to define antigen specificity and functional activity.

59 BASIC BIOLOGICAL SCIENCES↗