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Results for “membrane proteins”

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 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
NASA NTRS1989

Purification and identification of the fusicoccin binding protein from oat root plasma membrane

Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

NASA Discipline Number 29-20↗
DOE OSTI2021

The RIT1 C-terminus associates with lipid bilayers via charge complementarity

RIT1 is a member of the Ras superfamily of small GTPases involved in regulation of cellular signaling. Mutations to RIT1 are involved in cancer and developmental disorders. Like many Ras subfamily members, RIT1 is localized to the plasma membrane. However, RIT1 lacks the C-terminal prenylation that helps many other subfamily members adhere to cellular membranes. For this study, we used molecular dynamics simulations to examine the mechanisms by which the C-terminal peptide (CTP) of RIT1 associates with lipid bilayers. We show that the CTP is unstructured and that its membrane interactions depend on lipid composition. While a 12-residue region of the CTP binds strongly to anionic bilayers containing phosphatidylserine lipids, the CTP termini fray from the membrane allowing for accommodation of the RIT1 globular domain at the membrane-water interface.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1989

The sensory transduction pathways in bacterial chemotaxis

Bacterial chemotaxis is a useful model for investigating in molecular detail the behavioral response of cells to changes in their environment. Peritrichously flagellated bacteria such as coli and typhimurium swim by rotating helical flagella in a counterclockwise direction. If flagellar rotation is briefly reversed, the bacteria tumble and change the direction of swimming. The bacteria continuously sample the environment and use a temporal sensing mechanism to compare the present and immediate past environments. Bacteria respond to a broad range of stimuli including changes in temperature, oxygen concentration, pH and osmotic strength. Bacteria are attracted to potential sources of nutrition such as sugars and amino acids and are repelled by other chemicals. In the methylation-dependent pathways for sensory transduction and adaptation in E. coli and S. typhimurium, chemoeffectors bind to transducing proteins that span the plasma membrane. The transducing proteins are postulated to control the rate of autophosphorylation of the CheA protein, which in turn phosphorylates the CheY protein. The phospho-CheY protein binds to the switch on the flagellar motor and is the signal for clockwise rotation of the motor. Adaptation to an attractant is achieved by increasing methylation of the transducing protein until the attractant stimulus is cancelled. Responses to oxygen and certain sugars involve methylation-independent pathways in which adaption occurs without methylation of a transducing protein. Taxis toward oxygen is mediated by the electron transport system and changes in the proton motive force. Recent studies have shown that the methylation-independent pathway converges with the methylation-dependent pathway at or before the CheA protein.

Taylor, Barry L.↗
DOE OSTI2021

Maturation of Rhodobacter capsulatus Multicopper Oxidase CutO Depends on the CopA Copper Efflux Pathway and Requires the cutF Product (Final Report)

The goal is to achieve a comprehensive understanding of the nature, mechanism of function and biogenesis of c-type cytochromes and their oligomeric complexes. These cytochrome are ubiquitous heme proteins that are required for cellular bioenergetics, including photosynthesis (Ps) and respiration (Res), which are central to chemical energy (ATP) production by oxidative- and photophosphorylation. We contribute to the molecular characterization of individual and membrane-integral cytochromes as parts of Ps and Res electron transport (ET) pathways, using the facultative phototrophic bacteria of as excellent models for multidisciplinary investigations. Supported by this grant we discovered novel membrane-bound cytochromes and cyts c complexes that are important for Ps- and Res-ET. We worked out their biogenesis including maturation (covalent heme addition) and assembly of mature subunits into complexes. These involve fundamental process found virtually in all organisms and are fundamental processes with major significance to the “Photosynthetic Systems” program of DOE-BES, because the cytochromes are integral components of natural energy production systems. Elucidation of basic principles, and the knowledge gained by their study greatly impact on our ability to design, engineer, and create new natural, bio-hybrid, bio-inspired and artificial systems to better capture light, transduce and store its energy with improved efficacy and are of broad significance.

14 SOLAR ENERGY↗
NASA NTRS2006

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Rodriquez-Gomez, D.↗
DOE OSTI2023

Protein-activated atomic layer deposition for robust crude-oil-repellent hierarchical nano-armored membranes

Atomic layer deposition (ALD) offers unique capabilities to fabricate atomically engineered porous materials with precise pore tuning and multi-functionalization for diverse applications like advanced membrane separations towards sustainable energy-water systems. However, current ALD technique is inhibited on most non-polar polymeric membranes due to lack of accessible nucleation sites. Here, in this study, we report a facile method to efficiently promote ALD coating on hydrophobic surface of polymeric membranes via novel protein activation/sensitization. As a proof of concept, TiO 2 ALD-coated membranes activated by bovine serum albumin exhibit remarkable superhydrophilicity, ultralow underwater crude oil adhesion, and robust tolerance to rigorous environments including acid, alkali, saline, and ethanol. Most importantly, excellent cyclable crude oil-in-water emulsion separation performance can be achieved. The mechanism for activation/sensitization is rooted in reactivity for a particular set of amino acids. Furthermore, the universality of protein-sensitized ALD is demonstrated using common egg white, promising numerous potential usages in biomedical engineering, environmental remediation, lowcarbon manufacturing, catalysis, and beyond.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Dense, continuous membrane labeling and expansion microscopy visualization of ultrastructure in tissues

Abstract Lipid membranes are key to the nanoscale compartmentalization of biological systems, but fluorescent visualization of them in intact tissues, with nanoscale precision, is challenging to do with high labeling density. Here, we report ultrastructural membrane expansion microscopy (umExM), which combines an innovative membrane label and optimized expansion microscopy protocol, to support dense labeling of membranes in tissues for nanoscale visualization. We validate the high signal-to-background ratio, and uniformity and continuity, of umExM membrane labeling in brain slices, which supports the imaging of membranes and proteins at a resolution of ~60 nm on a confocal microscope. We demonstrate the utility of umExM for the segmentation and tracing of neuronal processes, such as axons, in mouse brain tissue. Combining umExM with optical fluctuation imaging, or iterating the expansion process, yields ~35 nm resolution imaging, pointing towards the potential for electron microscopy resolution visualization of brain membranes on ordinary light microscopes.

Science & Technology - Other Topics↗
DOE OSTI2020

Membrane stiffness and myelin basic protein binding strength as molecular origin of multiple sclerosis

Myelin basic protein (MBP) and its interaction with lipids of the myelin sheath plays an important part in the pathology of multiple sclerosis (MS). Previous studies observed that changes in the myelin lipid composition lead to instabilities and enhanced local curvature of MBP-lipid multilayer structures. We investigated the molecular origin of the instability and found that the diseased lipid membrane has a 25% lower bending rigidity, thus destabilizing smooth >1µm curvature radius structures such as in giant unilamellar vesicles. MBP-mediated assembling of lipid bilayers proceeds in two steps, with a slow second step occurring over many days where native lipid membranes assemble into well-defined multilayer structures, whereas diseased lipid membranes form folded assemblies with high local curvature. For both native and diseased lipid mixtures we find that MBP forms dense liquid phases on top of the lipid membranes mediating attractive membrane interactions. Furthermore, we observe MBP to insert into its bilayer leaflet side in case of the diseased lipid mixture, whereas there is no insertion for the native mixture. Insertion increases the local membrane curvature, and could be caused by a decrease of the sphingomyelin content of the diseased lipid mixture. These findings can help to open a pathway to remyelination strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

MOMP telonanoparticles, and related compositions, methods and systems

A telodendrimer-nanolipoprotein particle (t-NLP), comprising one or more membrane forming lipids, one or more telodendrimers, and a scaffold protein and a Chlamydia major outer membrane protein (MOMP) comprising a MOMP hydrophobic region, and related compositions methods and systems.

Coleman, Matthew A.↗
DOE OSTI2023

Solar water splitting Pt-nanoparticle photosystem I thylakoid systems: Catalyst identification, location and oligomeric structure

In this study, photosynthetic conversion of light energy into chemical energy occurs in sheet-like membrane-bound compartments called thylakoids and is mediated by large integral membrane protein-pigment complexes called reaction centers (RCs). Oxygenic photosynthesis of higher plants, cyanobacteria and algae requires the symbiotic linking of two RCs, photosystem II (PSII) and photosystem I (PSI), to split water and assimilate carbon dioxide. Worldwide there is a large research investment in developing RC-based hybrids that utilize the highly evolved solar energy conversion capabilities of RCs to power catalytic reactions for solar fuel generation. Of particular interest is the solar-powered production of H 2 , a clean and renewable energy source that can replace carbonbased fossil fuels and help provide for ever-increasing global energy demands. Recently, we developed thylakoid membrane hybrids with abiotic catalysts and demonstrated that photosynthetic Z-scheme electron flow from the light-driven water oxidation at PSII can drive H 2 production from PSI. One of these hybrid systems was created by self-assembling Pt-nanoparticles (PtNPs) with the stromal subunits of PSI that extend beyond the membrane plane in both spinach and cyanobacterial thylakoids. Using PtNPs as site-specific probe molecules, we report the electron microscopic (EM) imaging of oligomeric structure, location and organization of PSI in thylakoid membranes and provide the first direct visualization of photosynthetic Z-scheme solar water-splitting biohybrids for clean H 2 production.

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

Biomolecular complex viewed by dynamic nuclear polarization solid-state NMR spectroscopy

Solid-state nuclear magnetic resonance (ssNMR) is an indispensable tool for elucidating the structure and dynamics of insoluble and non-crystalline biomolecules. The recent advances in the sensitivity-enhancing technique magic-angle spinning dynamic nuclear polarization (MAS-DNP) have substantially expanded the territory of ssNMR investigations and enabled the detection of polymer interfaces in a cellular environment. This article highlights the emerging MAS-DNP approaches and their applications to the analysis of biomolecular composites and intact cells to determine the folding pathway and ligand binding of proteins, the structural polymorphism of low-populated biopolymers, as well as the physical interactions between carbohydrates, proteins, and lignin. Furthermore, these structural features provide an atomic-level understanding of many cellular processes, promoting the development of better biomaterials and inhibitors. It is anticipated that the capabilities of MAS-DNP in biomolecular and biomaterial research will be further enlarged by the rapid development of instrumentation and methodology.

36 MATERIALS SCIENCE↗
DOE OSTI2020

A mycobacterial ABC transporter mediates the uptake of hydrophilic compounds

Mycobacterium tuberculosis (Mtb) is an obligate human pathogen and the causative agent of tuberculosis. Although Mtb can synthesize vitamin B 12 (cobalamin) de novo, uptake of cobalamin has been linked to pathogenesis of tuberculosis. Mtb does not encode any characterized cobalamin transporter; however, the gene rv1819c was found to be essential for uptake of cobalamin. This result is difficult to reconcile with the original annotation of Rv1819c as a protein implicated in the transport of antimicrobial peptides such as bleomycin. In addition, uptake of cobalamin seems inconsistent with the amino acid sequence, which suggests that Rv1819c has a bacterial ATP-binding cassette (ABC)-exporter fold. In this paper, we present structures of Rv1819c, which reveal that the protein indeed contains the ABC-exporter fold, as well as a large water-filled cavity of about 7,700 Å, which enables the protein to transport the unrelated hydrophilic compounds bleomycin and cobalamin. On the basis of these structures, we propose that Rv1819c is a multi-solute transporter for hydrophilic molecules, analogous to the multidrug exporters of the ABC transporter family, which pump out structurally diverse hydrophobic compounds from cells.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Toll-like receptor 4-positive macrophages protect mice from Pasteurella pneumotropica-induced pneumonia

This study investigates Toll-like receptor 4 (TLR4)-positive macrophages in early recognition and clearance of pulmonary bacteria. TLR4 is a trans-membrane receptor that is the primary recognition molecule for lipopolysaccharide of gram-negative bacteria. The TLR4(Lps-del) mouse strains C57BL10/ScN (B10) and STOCK Abb(tm1) TLR4(Lps-del) Slc11a1(s)(B10 x C2D) are susceptible to pulmonary infections and develop pneumonia when naturally or experimentally infected by the opportunistic bacterium Pasteurella pneumotropica. Since these mice have the TLR4(Lps-del) genotype, we hypothesized that reconstitution of mice with TLR4-positive macrophages would provide resistance to this bacterium. A cultured macrophage cell line (C2D macrophages) and bone marrow cells from C2D mice were adoptively transferred to B10 and B10 x C2D mice by intraperitoneal injection. C2D macrophages increased B10 and B10 x C2D mouse resistance to P. pneumotropica. In C2D-recipient mice there was earlier transcription of tumor necrosis factor alpha and chemokines JE and macrophage inflammatory protein 2 (MIP-2) in the lungs of B10 and B10 x C2D mice, and there was earlier transcription of KC and MIP-1alpha in B10 x C2D mice. In addition, the course of inflammation following experimental Pasteurella challenge was altered in C2D recipients. C2D macrophages also protected B10 x C2D mice, which lack CD4(+) T cells. These data indicate that macrophages are critical for pulmonary immunity and can provide host resistance to P. pneumotropica. This study indicates that TLR4-positive macrophages are important for early recognition and clearance of pulmonary bacterial infections.

Non-NASA Center↗
DOE OSTI2021

CHARMM-GUI Drude prepper for molecular dynamics simulation using the classical Drude polarizable force field

Explicit treatment of electronic polarizability in empirical force fields (FFs) represents an extension over a traditional additive or pairwise FF and provides a more realistic model of the variations in electronic structure in condensed phase, macromolecular simulations. To facilitate utilization of the polarizable FF based on the classical Drude oscillator model, Drude Prepper has been developed in CHARMM-GUI. Drude Prepper ingests additive CHARMM protein structures file (PSF) and pre-equilibrated coordinates in CHARMM, PDB, or NAMD format, from which the molecular components of the system are identified. These include all residues and patches connecting those residues along with water, ions, and other solute molecules. This information is then used to construct the Drude FF-based PSF using molecular generation capabilities in CHARMM, followed by minimization and equilibration. In addition, inputs are generated for molecular dynamics (MD) simulations using CHARMM, GROMACS, NAMD, and OpenMM. Validation of the Drude Prepper protocol and inputs is performed through conversion and MD simulations of various heterogeneous systems that include proteins, nucleic acids, lipids, polysaccharides, and atomic ions using the aforementioned simulation packages. Stable simulations are obtained in all studied systems, including 5 μs simulation of ubiquitin, verifying the integrity of the generated Drude PSFs. Additionally, the ability of the Drude FF to model variations in electronic structure is shown through dipole moment analysis in selected systems. Finally, the capabilities and availability of Drude Prepper in CHARMM-GUI is anticipated to greatly facilitate the application of the Drude FF to a range of condensed phase, macromolecular systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

New molecular components of high and low affinity iron import systems in Drosophila

The high abundance and molecular versatility of iron have led to its universal presence in biological systems, yet its absorption is exceptionally challenging. Animals and yeasts use divalent metal transporters to import iron, but yeasts also employ the multicopper oxidase Fet3p for high-affinity iron uptake when iron-starved. Using long-term iron depletion in Drosophila, we identified four components involved in iron absorption: Multicopper oxidase-4 (Mco4), a Fet3p ortholog, is essential for surviving iron starvation, whereas the cytochrome b561 enzymes Fire (Ferric Iron Reductase) and Fire-like, as well as cytochrome b5 protein Firewood, are required for iron absorption under normal conditions. This study reports the presence of a high-affinity iron uptake system in an animal, a cytochrome b5 electron donor for ferric iron reduction, and intestinal ferric reductases, and provides a valuable resource for further exploration of genes involved in iron homeostasis, transport, and absorption.

Iron↗
DOE OSTI2022

Novel reusable animal model for comparative evaluation of in vivo growth and protein-expression of Escherichia coli O157 strains in the bovine rumen

Previously, we had demonstrated that Escherichia coli O157:H7 (O157) strain 86–24 expresses proteins involved in survival rather than virulence in vitro in rumen fluid from dairy cattle limit fed a maintenance diet. Here, we verified if this observation would be true for different O157 strains grown in vitro in rumen fluid from, and in vivo in the rumen of, animals on contrasting maintenance (high fiber) and lactation (high energy-protein) diets usually limit fed to dairy cattle. For the in vivo studies, an economical, novel, reusable and non-terminal rumen-fistulated animal model permitting simultaneous evaluation of multiple bacterial strains in the bovine rumen was developed. All experiments were conducted in duplicate using different animals to account for host-related variations. The O157 strains included, 86–24, EDL933 and the super shed SS-17. E. coli Nal R (#5735), derived from a bovine intestinal commensal E. coli, was included as a control. As expected, diet influenced ruminal pH and volatile fatty acid (VFA) composition. The pH ranged from 6.2–7.0 and total VFA concentrations from 109–141 μM/ml, in animals fed the maintenance diet. In comparison, animals fed the lactation diet had a ruminal pH ranging between 5.18–6.0, and total VFA of 125–219 μM/ml. Strain dependent differences in O157 recovery from the rumen fluid of cattle fed either diet was observed, both in vitro and in vivo, with O157 strains 86–24 and EDL933 demonstrating similar survival patterns. Analysis of the O157 proteomes expressed in the rumen fluid/rumen verified previous observations of adaptive responses. Any difference in the adaptive response was mainly influenced by the animal’s diet and growth conditions (in vitro and in vivo) and not the O157 strain. These new insights into the O157 responses could help formulate modalities to control O157 across strains in cattle at all stages of husbandry.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Computing Potential of the Mean Force Profiles for Ion Permeation Through Channelrhodopsin Chimera, C1C2

Umbrella sampling, coupled with a weighted histogram analysis method (US-WHAM), can be used to construct potentials of mean force (PMFs) for studying the complex ion permeation pathways of membrane transport proteins. Despite the widespread use of US-WHAM, obtaining a physically meaningful PMF can be challenging. Here, we provide a protocol to resolve that issue. Then, we apply that protocol to compute a meaningful PMF for sodium ion permeation through channelrhodopsin chimera, C1C2, for illustration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

D-Band EPR and ENDOR Spectroscopy of 15 N-Labeled Photosystem I

For billions of years, nature has optimized the photosynthetic machinery that converts light energy into chemical energy. Key primary reactions of photosynthesis occur in large membrane protein-cofactor complexes. The light-induced sequential electron transfer reactions occur through a chain of donor/acceptor cofactors embedded in the protein matrix resulting in a long-lived transmembrane charge-separated state. EPR is the method of choice to study electron transfer and the interaction of protein environment with redox-active cofactors. However, the spectra of organic cofactor radicals typically are not fully resolved and severely overlap at conventional X-band EPR. Even at Q-band EPR, this overlap is present and often a serious problem. As a result, there is a large variation of the reported EPR data and limited understanding of electronic structures of several redox-active cofactors. These serious problems can often be overcome by the excellent spectral resolution provided by high-frequency EPR (HF EPR). In this work, we study the electronic structure of the primary electron donor P 700 and the secondary electron acceptor A 1 of Photosystem I (PSI) using 130 GHz (D-band) EPR and Electron-Nuclear-Double-Resonance (ENDOR) spectroscopy. PSI was isotopically labeled with 15 N (I = $\frac{1/2}$) to avoid quadrupolar interactions in the most abundant nitrogen isotope 14 N (I = 1) and simplify the ENDOR spectra. ENDOR spectroscopy is central for determining the hyperfine coupling of nitrogen atoms of the two chlorophyll molecules comprising oxidized P 700 and the involvement of protein nitrogen atoms with reduced A 1 . While HF ENDOR of A 1 - allows identification of two nitrogen atoms, HF ENDOR of P 700 + still does not permit unique assignment of the recorded hyperfine couplings.

15N↗