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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 577 records · Page 32
All sourcesNASA NTRSDOE OSTI
DOE OSTI2017

Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst

Microbial conversion of methane to high-value bio-based chemicals and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO 2 lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense , encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. As a result, given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.

09 BIOMASS FUELS↗
DOE OSTI2017

Reversible Lifting of Surface Supported Lipid Bilayers with a Membrane-Spanning Nonionic Triblock Copolymer

Neutron reflectometry was used to monitor structural variations in surface supported DMPC bilayers induced by the addition of Triton X-100, a surfactant commonly used to aid solubilization of membrane proteins, and the co-addition of a membrane spanning non-ionic amphiphilic triblock copolymer, (PEO 117 -PPO 47 -PE O117 , Pluronic F98). Surfactant addition causes slight compression of the bilayer thickness and the creation of a distinct EO layer that increases the hydrophilic layer proximal to the supporting substrate (i.e., a water and EO gap between the lipid bilayer and quartz) to 6.8 ± 0.4 Å. Addition of the triblock copolymer into the DMPC: Triton X-100 bilayer increases the complexity (broadens) the lipid phase transition, further compresses the bilayer, and continues to expand the proximal hydrophilic layer thickness. The observed structural changes are temperature dependent with transmembrane polymer insertion achieved at 37 °C leading to a compressed membrane thickness of 39.2 ± 0.2 Å and proximal gap of 45.2 ± 0.2 Å. Temperature driven exclusion of the polymer at 15 °C causes partitioning of the polymer into the proximal space generating a large hydrogel cushion 162 ± 16 Å thick. An intermediate gap width (10 – 27 Å) is achieved at room temperature (22 – 25 °C). The temperature-driven changes in the proximal hydrophilic gap dimensions are shown to be reversible but thermal history causes variation in magnitude. Temperature-driven changes in polymer association with a supported lipid bilayer offer a facile means to reversibly control both the membrane characteristics as well as the separation between membrane and solid substrate.

36 MATERIALS SCIENCE↗
DOE OSTI2017

A Computational Approach for Modeling Neutron Scattering Data from Lipid Bilayers

Biological cell membranes are responsible for a range of structural and dynamical phenomena crucial to a cell's well-being and its associated functions. Due to the complexity of cell membranes, lipid bilayer systems are often used as biomimetic models. These systems have led to signficant insights into vital membrane phenomena such as domain formation, passive permeation and protein insertion. Experimental observations of membrane structure and dynamics are, however, limited in resolution, both spatially and temporally. Importantly, computer simulations are starting to play a more prominent role in interpreting experimental results, enabling a molecular under- standing of lipid membranes. Particularly, the synergy between scattering experiments and simulations offers opportunities for new discoveries in membrane physics, as the length and time scales probed by molecular dynamics (MD) simulations parallel those of experiments. We also describe a coarse-grained MD simulation approach that mimics neutron scattering data from large unilamellar lipid vesicles over a range of bilayer rigidity. Specfically, we simulate vesicle form factors and membrane thickness fluctuations determined from small angle neutron scattering (SANS) and neutron spin echo (NSE) experiments, respectively. Our simulations accurately reproduce trends from experiments and lay the groundwork for investigations of more complex membrane systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Reparameterizing a Lipid Force Field Using Small-Angle X-ray Scattering to Improve Predictions of Multicomponent Membranes under Organic Solvent Stress

Understanding and predicting properties of lipid-bilayer membranes are essential to elucidating organismal physiology and pathophysiology. Therefore, substantial efforts have been undertaken to derive accurate molecular-mechanics force fields (FF) to allow simulation of their properties; however, much of these past efforts focused on tuning force fields to accurately reproduce the properties of model single-component membranes and not multicomponent or stressed membranes. Here, we tuned the CHARMM36 FF for a simple 2-component model of a Gram-positive bacterium. This updated force field is parametrized against a single condensed-phase property, the small-angle X-ray scattering (SAXS) intensities of the 2-component lipid system, using a version of ForceBalance implemented previously (named ForceBalance-SAS) with SAXS and small-angle neutron scattering (SANS) intensities as optimization targets. After tuning, we observe improved agreement with the experimental SAXS of the membrane under 1-butanol and tetrahydrofuran solvent stresses, with a factor of 10.0 and 7.5 reduction, respectively, in χ 2 , the measure of the discrepancy between experimental and computed SAXS intensities. Furthermore, this reparametrized force field yielded improved agreement between experimental and simulated membrane thicknesses for the pure lipid systems. However, we note limitations in transferability and diagnose the source of such limitations, particularly the need for SANS in addition to SAXS.

Demerdash, Omar N. A. [Oak Ridge National Laborato↗
DOE OSTI2016

Dynamics of crowding-induced mixing in phase separated lipid bilayers

We use fluorescence microscopy to examine the dynamics of the crowding-induced mixing transition of liquid ordered (L o )–liquid disordered (L d ) phase separated lipid bilayers when the following particles of increasing size bind to either the L o or L d phase: Ubiquitin, green fluorescent protein (GFP), and nanolipoprotein particles (NLPs) of two diameters. These proteinaceous particles contained histidine-tags, which were phase targeted by binding to iminodiacetic acid (IDA) head groups, via a Cu 2+ chelating mechanism, of lipids that specifically partition into either the Lo phase or Ld phase. The degree of steric pressure was controlled by varying the size of the bound particle (10–240 kDa) and the amount of binding sites present (i.e., DPIDA concentrations of 9 and 12 mol%) in the supported lipid multibilayer platform used here. We develop a mass transfer-based diffusional model to analyze the observed L o phase domain dissolution that, along with visual observations and activation energy calculations, provides insight into the sequence of events in crowding-induced mixing. Furthermore, our results suggest that the degree of steric pressure and target phase influence not only the efficacy of steric-pressure induced mixing, but the rate and controlling mechanism for which it occurs.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Energetic and dynamic analysis of transport of Na + and K + through a cyclic peptide nanotube in water and in lipid bilayers

Potential of mean force (PMF) profiles and position-dependent diffusion coefficients of Na + and K + are calculated to elucidate the translocation of ions through a cyclic peptide nanotube, composed of 8 × cyclo[-(D-Leu-Trp) 4- ] rings, in water and in hydrated DMPC bilayers. The PMF profiles and PMF decomposition analysis for the monovalent cations show that favorable interactions of the cations with the CPN as well as the lipid bilayer and dehydration free energy penalties are two major competing factors which determine the free energy surface for ion transport through CPNs both in water and lipid bilayers, and that the selectivity of CPNs to cations mainly arises from favorable interaction energies of cations with CPNs and lipid bilayers that are more dominant than the dehydration penalties. Calculations of the position-dependent diffusion coefficients and dynamic friction kernels of the cations indicate that the dehydration process along with the molecular rearrangements occurring outside the channel and the coupling of the ion motions with the chain-structured water movements inside the channel lead to decrease of the diffusion coefficients far away from the channel entrance and also reduced coefficients inside the channel. Here the PMF and diffusivity profiles for Na + and K + reveal that the energetics of ion transport through the CPN are governed by global interactions of ions with all the components in the system while the diffusivity of ions through the channel is mostly determined by local interactions of ions with the confined water molecules inside the channel. Comparison of Na + and K + ion distributions based on overdamped Brownian dynamics simulations based on the PMF and diffusivity profiles with the corresponding results from molecular dynamics shows good agreement, indicating accuracy of the Bayesian inference method for determining diffusion coefficients in this application. In addition this work shows that position-dependent diffusion coefficients of ions are required to explain the dynamics and conductance of ions through the CPN properly.

36 MATERIALS SCIENCE↗
DOE OSTI2017

Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers

OprF has a central role in Pseudomonas aeruginosa virulence and thus provides a putative target for either vaccines or antibiotic cofactors that could overcome the bacterium’s natural resistance to antibiotics. Here we describe a procedure to optimize the production of highly pure and functional OprF porins that are then incorporated into a tethered lipid bilayer. This is a stable biomimetic system that provides the capability to investigate structural aspects and function of OprF using and neutron reflectometry and electrical impedance spectroscopy. The recombinant OprF produced using the optimized cell-free procedure yielded a quantity of between 0.5 to 1.0 mg/mL with a purity ranging from 85 to 91% in the proteoliposomes. The recombinant OprF is capable of binding IFN-γ and is correctly folded in the proteoliposomes. Because OprF proteins form pores the biomimetic system allowed the measurement of OprF conductance using impedance spectroscopy. The neutron reflectometry measurements showed that the OprF protein is incorporated into the lipid bilayer but with parts of the protein in both the regions above and below the lipid bilayer. Those structural aspects are coherent with the current assumed structure of a transmembrane N-terminal domain composed by eight stranded beta-barrels and a globular C-terminal domain located in the periplasm. Currently there are no crystal structures available for OprF. The experimental model system that we describe provides a basis for further fundamental studies of OprF and particularly for the ongoing biotechnological development of OprF as a target for antibacterial drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature

We report biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb 3 . This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb 3 carbohydrate and ceramide moieties modulated the toxin’s capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

36 MATERIALS SCIENCE↗
DOE OSTI2018

Lipid binding promotes the open conformation and tumor-suppressive activity of neurofibromin 2

Neurofibromatosis type 2 (NF2) is a tumor-forming disease of the nervous system caused by deletion or by loss-of-function mutations in NF2, encoding the tumor suppressing protein neurofibromin 2 (also known as schwannomin or merlin). Neurofibromin 2 is a member of the ezrin, radixin, moesin (ERM) family of proteins regulating the cytoskeleton and cell signaling. The correlation of the tumor-suppressive function and conformation (open or closed) of neurofibromin 2 has been subject to much speculation, often based on extrapolation from other ERM proteins, and controversy. Here we show that lipid binding results in the open conformation of neurofibromin 2 and that lipid binding is necessary for inhibiting cell proliferation. Collectively, our results provide a mechanism in which the open conformation is unambiguously correlated with lipid binding and localization to the membrane, which are critical for the tumor-suppressive function of neurofibromin 2, thus finally reconciling the long-standing conformation and function debate.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural characterization of life-extending Caenorhabditis elegans Lipid Binding Protein 8

The lysosome plays a crucial role in the regulation of longevity. Lysosomal degradation is tightly coupled with autophagy that is induced by many longevity paradigms and required for lifespan extension. The lysosome also serves as a hub for signal transduction and regulates longevity via affecting nuclear transcription. One lysosome-to-nucleus retrograde signaling pathway is mediated by a lysosome-associated fatty acid binding protein LBP-8 in Caenorhabditis elegans. LBP-8 shuttles lysosomal lipids into the nucleus to activate lipid regulated nuclear receptors NHR-49 and NHR-80 and consequently promote longevity. However, the structural basis of LBP-8 action remains unclear. Here, we determined the first 1.3 Å high-resolution structure of this life-extending protein LBP-8, which allowed us to identify a structurally conserved nuclear localization signal and amino acids involved in lipid binding. Additionally, we described the range of fatty acids LBP-8 is capable of binding and show that it binds to life-extending ligands in worms such as oleic acid and oleoylethanolamide with high affinity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The allosteric mechanism leading to an open-groove lipid conductive state of the TMEM16F scramblase

TMEM16F is a Ca 2+ -activated phospholipid scramblase in the TMEM16 family of membrane proteins. Unlike other TMEM16s exhibiting a membrane-exposed hydrophilic groove that serves as a translocation pathway for lipids, the experimentally determined structures of TMEM16F shows the groove in a closed conformation even under conditions of maximal scramblase activity. It is currently unknown if/how TMEM16F groove can open for lipid scrambling. Here we describe the analysis of ~400 µs all-atom molecular dynamics (MD) simulations of the TMEM16F revealing an allosteric mechanism leading to an open-groove, lipid scrambling competent state of the protein. The groove opens into a continuous hydrophilic conduit that is highly similar in structure to that seen in other activated scramblases. The allosteric pathway connects this opening to an observed destabilization of the Ca 2+ ion bound at the distal site near the dimer interface, to the dynamics of specific protein regions that produces the open-groove state to scramble phospholipids.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Efficient lipid extraction and quantification of fatty acids from algal biomass using accelerated solvent extraction (ASE)

Accelerated solvent extraction (ASE), a commercially available pressurized fluid extraction technique and conventional manual extraction were compared to identify the most effective chloroform–methanol extraction method for algal lipids. Using optimal ASE operating conditions (methanol/chloroform = 2 : 1 by vol, 100 °C, static time of 5 min, and four static cycles), the lipid contents of Chlorella vulgaris, C. sorokiniana, C. zofingiensis and Nannochloropsis gaditana were 27.5%, 25.8%, 15.2%, 29.8% of dry biomass, respectively. Here, the total fatty acid methyl ester (FAME) content of dry biomass from ASE extraction was found to be 1.3–2.7 fold higher than that from conventional manual extract from these species, demonstrating that ASE exhibited significant improvement for lipid and FAME recovery. Furthermore, ASE showed the capacity to extract all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA) as 3.0% of dry biomass from Nannochloropsis gaditana suggesting that ASE has the potential to obtain polyunsaturated fatty acids (PUFAs) as well.

09 BIOMASS FUELS↗
DOE OSTI2020

Decoupling copolymer, lipid and carbon nanotube interactions in hybrid, biomimetic vesicles

Bilayer vesicles that mimic a real biological cell can be tailored to carry out a specific function by manipulating the molecular composition of the amphiphiles. These bio-inspired and bio-mimetic structures are increasingly being employed for a number of applications from drug delivery to water purification and beyond. Complex hybrid bilayers are the key building blocks for fully synthetic vesicles that can mimic biological cell membranes, which often contain a wide variety of molecular species. While the assembly and morpholgy of pure phospholid bilayer vesicles is well understood, the functionality and structure dramaticlly changes when copolymer and/or carbon nanotube porins (CNTP) are added. The aim of this study is to understand how the collective molecular interactions within hybrid vesicles affect their nanoscale structure and properties. In situ small and wide angle X-ray scattering (SAXS/WAXS) and molecular dynamics simulations (MD) are used to investigate the morphological effect of molecular interactions between polybutadiene polyethylene oxide, lipids and carbon nanotubes (CNT) within the hybrid vesicle bilayer. Within the lipid/copolymer system, the hybrid bilayer morphology transitions from phase separated lipid and compressed copolymer at low copolymer loadings to a mixed bilayer where opposing lipids are mostly separated from the inner region. This transition begins between 60 wt% and 70 wt%, with full homogenization observed by 80 wt% copolymer. The incorporation of CNT into the hybrid vesicles increases the bilayer thickness and enhances the bilayer symmetry. Analysis of the WAXS and MD indicate that the CNT–dioleoyl interactions are much stronger than the CNT–polybutadiene.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structure and dynamics of lipid membranes interacting with antivirulence end-phosphorylated polyethylene glycol block copolymers

The structure and dynamics of lipid membranes in the presence of extracellular macromolecules are critical for cell membrane functions and many pharmaceutical applications. The pathogen virulence-suppressing end-phosphorylated polyethylene glycol (PEG) triblock copolymer (Pi-ABAPEG) markedly changes the interactions with lipid vesicle membranes and prevents PEG-induced vesicle phase separation in contrast to the unphosphorylated copolymer (ABAPEG). Pi-ABAPEG weakly absorbs on the surface of lipid vesicle membranes and slightly changes the structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar vesicles at 37 °C, as evidenced by small angle neutron scattering. X-ray reflectivity measurements confirm the weak adsorption of Pi-ABAPEG on DMPC monolayer, resulting in a more compact DMPC monolayer structure. Neutron spin-echo results show that the adsorption of Pi-ABAPEG on DMPC vesicle membranes increases the membrane bending modulus $κ$.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Mechanisms of lipid droplet biogenesis

Abstract Lipid droplets (LDs) are organelles that compartmentalize nonbilayer-forming lipids in the aqueous cytoplasm of cells. They are ubiquitous in most organisms, including in animals, protists, plants and microorganisms. In eukaryotes, LDs are believed to be derived by a budding and scission process from the surface of the endoplasmic reticulum, and this occurs concomitantly with the accumulation of neutral lipids, most often triacylglycerols and steryl esters. Overall, the mechanisms underlying LD biogenesis are difficult to generalize, in part because of the involvement of different sets of both evolutionarily conserved and organism-specific LD-packaging proteins. Here, we briefly compare and contrast these proteins and the allied processes responsible for LD biogenesis in cells of animals, yeasts and plants.

Biochemistry & Molecular Biology↗
DOE OSTI2019

MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure of Mycobacterium smegmatis MmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Cyclization of archaeal membrane lipids impacts membrane protein activity and archaellum formation

Enhancement of the cyclization of membrane lipids GDGTs (glycerol dialkyl glycerol tetraethers) is a critical strategy for archaea to adapt to various environmental stresses. However, the physiological function of membrane lipid cyclization remains unclear. Here, we reported that the GDGT ring synthases mutant, deficient in GDGT cyclization, inhibited archaellum formation and reduced cell motility in thermoacidophilic crenarchaeon Sulfolobus acidocaldarius . This inhibition was caused by decreased transcription of the archaellum operon, likely due to cleavage of the C-terminal domains in transmembrane proteins ArnRs, the transcription factors that regulate archaellum operon expression. The transcriptomic and proteomic analysis showed deficiency of GDGT cyclization broadly impacted the expression of membrane associate proteins, including respiratory chain proteins, and decreased cellular ATP concentration. Moreover, phylogenetic analysis demonstrated that the correlation between GDGT cyclization and archaellum formation is widespread among (hyper)thermophilic archaea, and this was further verified in the euryarchaeon Thermococcus kodakarensis. Our findings suggested that archaea modify their membrane lipids to profoundly alter cellular appendages and cell physiology to adapt to environmental fluctuations.

Yang, Wei (ORCID:0000000262755981)↗
DOE OSTI2016

Conformational heterogeneity in closed and open states of the KcsA potassium channel in lipid bicelles

The process of ion channel gating—opening and closing—involves local and global structural changes in the channel in response to external stimuli. Conformational changes depend on the energetic landscape that underlies the transition between closed and open states, which plays a key role in ion channel gating. For the prokaryotic, pH-gated potassium channel KcsA, closed and open states have been extensively studied using structural and functional methods, but the dynamics within each of these functional states as well as the transition between them is not as well understood. In this study, we used solution nuclear magnetic resonance (NMR) spectroscopy to investigate the conformational transitions within specific functional states of KcsA. We incorporated KcsA channels into lipid bicelles and stabilized them into a closed state by using either phosphatidylcholine lipids, known to favor the closed channel, or mutations designed to trap the channel shut by disulfide cross-linking. A distinct state, consistent with an open channel, was uncovered by the addition of cardiolipin lipids. Using selective amino acid labeling at locations within the channel that are known to move during gating, we observed at least two different slowly interconverting conformational states for both closed and open channels. The pH dependence of these conformations and the predictable disruptions to this dependence observed in mutant channels with altered pH sensing highlight the importance of conformational heterogeneity for KcsA gating.

60 APPLIED LIFE SCIENCES↗