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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 OSTI2023

Resolving metal binding properties within subunits of a multimeric enzyme Mnx by surface induced dissociation and native ion mobility mass spectrometry

Multi-subunit enzymes function as coordinated assemblies. Yet most enzymatic assays measure the summed output of all populations in solution and cannot easily differentiate contributions of individual subunits. Native mass spectrometry detects intact protein complexes in the gas phase. Surface induced dissociation further releases subunits from protein complexes while retaining compact conformations and bound ligands. Combined with ion mobility, the released subunits can then be carefully monitored for more in-depth structural analysis. Mnx is a unique bacterial multicopper oxidase complex that oxidizes Mn(II) to form MnO 2 minerals, and is composed of three subunits: MnxG, a multicopper oxidase containing the active site, and two accessory proteins, MnxE and MnxF which also bind copper ions. Other known multicopper oxidases do not require accessory proteins, therefore the functions of MnxE and MnxF are not well understood. Here, we use native mass spectrometry with surface induced dissociation and ion mobility to characterize the metal binding properties of Mnx with two metals, catalytic Cu(II) and Mn(II) substrate. We demonstrate our assay can detect subtle structural changes within each subunit, which are presumably related to the allosteric mechanism. We also noticed that ionic strength and solution composition can impact metal binding and must be carefully investigated for such experiments.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes

Fatty acid (FA) transfer proteins extract FA from membranes and sequester them to facilitate their movement through the cytosol. Detailed structural information is available for these soluble protein–FA complexes, but the structure of the protein conformation responsible for FA exchange at the membrane is unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we define the conformational change from a “closed” FakB1 state to an “open” state that associates with the membrane and provides a path for entry and egress of the FA. Using NMR spectroscopy, we identified a conformationally flexible dynamic region in FakB1, and X-ray crystallography of FakB1 mutants captured the conformation of the open state. In addition, molecular dynamics simulations show that the new amphipathic α-helix formed in the open state inserts below the phosphate plane of the bilayer to create a diffusion channel for the hydrophobic FA tail to access the hydrocarbon core and place the carboxyl group at the phosphate layer. The membrane binding and catalytic properties of site-directed mutants were consistent with the proposed membrane docked structure predicted by our molecular dynamics simulations. Finally, the structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a conceptual framework for how these proteins interact with the membrane to create a diffusion channel from the FA location in the bilayer to the protein interior.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with K D values in the low millimolar range. Since these K D values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

La 3+ and Y 3+ interactions with the carboxylic acid moiety at the liquid/vapor interface: Identification of binding complexes, charge reversal, and detection limits

Specific interactions of yttrium and lanthanum ions with a fatty acid Langmuir monolayer were investigated using vibrational sum frequency spectroscopy. The trivalent ions were shown to interact with the charged form of the carboxylic acid group from nanomolar concentrations (<300 nM). Analysis of the spectral features from both the symmetric and the asymmetric carboxylate modes reveals the presence of at least three distinct coordination structures linked to specific binding configurations. Although the same species were identified for both La 3+ and Y 3+ , they display a different concentration dependence, highlighting the ion-specificity of the interaction. From the analysis of the response of interfacial water molecules, the reversal of the surface charge, as well as the formation of yttrium hydroxide complexes, were detected upon increasing the amount of salt in solution. The binding interaction and kinetics of absorption are sensitive to the solution pH, showing a distinct ion speciation in the interfacial region when compared to the bulk. Changing the subphase pH or adding a monovalent background electrolyte that promotes deprotonation of the carboxylic acid headgroup could further improve the detection limit of La 3+ and Y 3+ to concentrations < 100 nM. These findings demonstrate that nM concentrations of trace metals contaminants, typically found on monovalent salts, can significantly influence the binding structure and kinetics in Langmuir monolayers.

01 COAL, LIGNITE, AND PEAT↗
DOE OSTI2025

Substituent size versus metal binding of inhibitors with variants of influenza endonuclease

The influenza virus causes a significant burden of illness each year. Although vaccination is the most effective method to prevent seasonal influenza infection, viral escape mechanisms make vaccine composition difficult to predict. Antivirals are crucial for decreasing rates of morbidity and mortality from influenza viral infection. The newest anti-influenza drugs target the RNA-dependent RNA polymerase acidic N-terminal (PA N ) endonuclease, a critical component of influenza viral replication machinery. This study examines the structure of inhibitors of PAN that utilize a hydroxypyridinone-based metal-binding pharmacophore (MBP). Specifically, this report explores how the size of substituent groups impacts the binding conformation and affinity of a series of compounds against both wild-type (WT) and resistance mutant strains, I38T and E23K. Co-crystal structures revealed that the distance between compounds and enzyme residue 38 was conserved to maintain strong interactions, resulting in deviations from ideal coordination geometries at the active site metal centers. This suggests the interactions with residue 38 with each compound is important and can impact inhibitor potency as a consequence of distortions in the metal binding geometry of the compounds.

Endonuclease↗
DOE OSTI2020

Development of Density Functional Tight-Binding Parameters Using Relative Energy Fitting and Particle Swarm Optimization

We provide a strategy to optimize density functional tight-binding (DFTB) parameterization for the calculation of the structures and properties of organic molecules consisting of hydrogen, carbon, nitrogen, and oxygen. We utilize an objective function based on similarity measurements and the Particle Swarm Optimization (PSO) method to find an optimal set of parameters. This objective function considers not only the common DFTB descriptors of binding energies and atomic forces but also incorporates relative energies of isomers into the fitting procedure for more chemistry-driven results. The quality in the description of the binding energies and atomic forces is measured based on the Ballester similarity index and relative energies through a similarity index induced by the Levenshtein edit distance to quantify the correct energetic order of isomers. Training and testing datasets were created to include all relevant chemical functional groups. Finally, the accuracy of this strategy is assessed, and its range of applicability is discussed by comparison against our previous parameterization. The improved performance of the new DFTB parameterization is validated with respect to the density functional theory large datasets QM-9 and ANI-1, where excellent agreement is found between the structures and properties available in these datasets, and the ones obtained with DFTB.

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

Benchmarking DFT Accuracy in Predicting O 1s Binding Energies on Metals

X-ray photoelectron spectroscopy (XPS) is a powerful tool for probing the electronic structure and composition of materials, particularly metals and metal oxides of relevance to solar cells and catalysis. Density functional theory (DFT) is often used to support XPS peak assignments, but its reliability for predicting oxygen species is not well established. Here, we compile a large data set of experimental oxygen binding energies and evaluate corresponding DFT predictions. We find that as the binding energies of metal-bound atomic oxygen species increase, especially above ≈530 eV, there is a general decrease in the accuracy of DFTpredicted values. Thus, high-binding-energy atomic oxygen species, such as those proposed as active for selective Ag-catalyzed epoxidation, are less well represented. The chemical nature of the oxygen species also influences accuracy, with molecularly bound species more reliably captured across the entire range of energies. These findings illustrate the limitations of DFT for interpreting XPS spectra and provide a benchmark for improving computational methods.

Adsorption↗
DOE OSTI2026

A Chimeric LBT-GFP Biosensor Exhibits Antithetical Fluorescence Responses to Ca 2+ and Dy 3+ Binding

Rare earth elements (REEs) are critical components in emerging technologies, but their mining and refining processes are often laborious, costly, and environmentally damaging. Developing green and efficient separation methods for REEs is crucial. Biomolecular approaches using lanthanide-binding proteins and peptides show promise for selective REE extraction and separation. In this study, we present the design and characterization of a genetically encoded fluorescence indicator (GEFI) construct that combines a superfolder green fluorescent protein (sfGFP) with a dual lanthanide-binding tag (2×dLBT). The 2×dLBT insert induces conformational changes in sfGFP upon lanthanide binding, modulating the fluorescence intensity. The sfGFP-2×dLBT biosensor exhibited distinct fluorescence responses to different lanthanide ions, with the highest dynamic range observed for heavy REEs like dysprosium (Dy 3+ ). Interestingly, the sensor displayed an antithetical response, where low concentrations of lanthanides initially quenched the fluorescence, but higher concentrations led to a significant fluorescence increase (1.5-fold). The Ca 2+ ion on the other hand showed only a dose-dependent quenching of the fluorescence response. Based on these observations, the biphasic response of the biosensor to lanthanides was eliminated by pretreating the sensor with calcium, which further expanded the dynamic range up to 3-fold for Dy 3+ . The lanthanide-selective and concentration-dependent fluorescence changes of the sfGFP-2×dLBT biosensor demonstrate its potential as a platform for developing specific sensors for various REEs. These sensors could enable rapid and cost-effective determination of REE composition in complex mixtures, facilitating the separation and recovery of critical REEs from electronic waste and other REE-containing sources.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Binding of the substrate analog methanol in the oxygen-evolving complex of photosystem II in the D1-N87A genetic variant of cyanobacteria

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction is catalyzed at the Mn 4 Ca-oxo cluster in the oxygen-evolving complex (OEC), which cycles through five light-driven S-state intermediates (S 0 –S 4 ). A detailed mechanism of the reaction remains elusive as it requires knowledge of the delivery and binding of substrate water in the higher S-state intermediates. In this paper, we use two-dimensional (2D) hyperfine sublevel correlation spectroscopy, in conjunction with quantum mechanics/molecular mechanics (QM/MM) and density functional theory (DFT), to probe the binding of the substrate analog, methanol, in the S 2 state of the D1-N87A variant of PSII from Synechocystis sp. PCC 6803. The results indicate that the size and specificity of the “narrow” channel is altered in D1-N87A PSII, allowing for the binding of deprotonated 13 C-labeled methanol at the Mn4(IV) ion of the catalytic cluster in the S 2 state. This has important implications on the mechanistic models for water oxidation in PSII.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site

Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2002

Rationally designed fluorescently labeled sulfate-binding protein mutants: evaluation in the development of a sensing system for sulfate

Periplasmic binding proteins from E. coli undergo large conformational changes upon binding their respective ligands. By attaching a fluorescent probe at rationally selected unique sites on the protein, these conformational changes in the protein can be monitored by measuring the changes in fluorescence intensity of the probe which allow the development of reagentless sensing systems for their corresponding ligands. In this work, we evaluated several sites on bacterial periplasmic sulfate-binding protein (SBP) for attachment of a fluorescent probe and rationally designed a reagentless sensing system for sulfate. Eight different mutants of SBP were prepared by employing the polymerase chain reaction (PCR) to introduce a unique cysteine residue at a specific location on the protein. The sites Gly55, Ser90, Ser129, Ala140, Leu145, Ser171, Val181, and Gly186 were chosen for mutagenesis by studying the three-dimensional X-ray crystal structure of SBP. An environment-sensitive fluorescent probe (MDCC) was then attached site-specifically to the protein through the sulfhydryl group of the unique cysteine residue introduced. Each fluorescent probe-conjugated SBP mutant was characterized in terms of its fluorescence properties and Ser171 was determined to be the best site for the attachment of the fluorescent probe that would allow for the development of a reagentless sensing system for sulfate. Three different environment-sensitive fluorescent probes (1,5-IAEDANS, MDCC, and acylodan) were studied with the SBP171 mutant protein. A calibration curve for sulfate was constructed using the labeled protein and relating the change in the fluorescence intensity with the amount of sulfate present in the sample. The detection limit for sulfate was found to be in the submicromolar range using this system. The selectivity of the sensing system was demonstrated by evaluating its response to other anions. A fast and selective sensing system with detection limits for sulfate in the submicromolar range was developed. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 517-526, 2002.

Evaluation Studies↗
NASA NTRS1999

Apocalmodulin and Ca2+ calmodulin bind to the same region on the skeletal muscle Ca2+ release channel

The skeletal muscle Ca2+ release channel (RYR1) is regulated by calmodulin in both its Ca2+-free (apocalmodulin) and Ca2+-bound (Ca2+ calmodulin) states. Apocalmodulin is an activator of the channel, and Ca2+ calmodulin is an inhibitor of the channel. Both apocalmodulin and Ca2+ calmodulin binding sites on RYR1 are destroyed by a mild tryptic digestion of the sarcoplasmic reticulum membranes, but calmodulin (either form), bound to RYR1 prior to tryptic digestion, protects both the apocalmodulin and Ca2+ calmodulin sites from tryptic destruction. The protected sites are after arginines 3630 and 3637 on RYR1. These studies suggest that both Ca2+ calmodulin and apocalmodulin bind to the same or overlapping regions on RYR1 and block access of trypsin to sites at amino acids 3630 and 3637. This sequence is part of a predicted Ca2+ CaM binding site of amino acids 3614-3642 [Takeshima, H., et al. (1989) Nature 339, 439-445].

NASA Discipline Musculoskeletal↗
NASA NTRS1998

Calcium-binding proteins and development

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

Review↗
NASA NTRS1997

Reduced naphthylphthalamic acid binding in the tir3 mutant of Arabidopsis is associated with a reduction in polar auxin transport and diverse morphological defects

Polar auxin transport plays a key role in the regulation of plant growth and development. To identify genes involved in this process, we have developed a genetic procedure to screen for mutants of Arabidopsis that are altered in their response to auxin transport inhibitors. We recovered a total of 16 independent mutants that defined seven genes, called TRANSPORT INHIBITOR RESPONSE (TIR) genes. Recessive mutations in one of these genes, TIR3, result in altered responses to transport inhibitors, a reduction in polar auxin transport, and a variety of morphological defects that can be ascribed to changes in indole-3-acetic acid distribution. Most dramatically, tir3 seedlings are strongly deficient in lateral root production, a process that is known to depend on polar auxin transport from the shoot into the root. In addition, tir3 plants display a reduction in apical dominance as well as decreased elongation of siliques, pedicels, roots, and the inflorescence. Biochemical studies indicate that tir3 plants have a reduced number of N-1-naphthylphthalamic (NPA) binding sites, suggesting that the TIR3 gene is required for expression, localization, or stabilization of the NPA binding protein (NBP). Alternatively, the TIR3 gene may encode the NBP. Because the tir3 mutants have a substantial defect in NPA binding, their phenotype provides genetic evidence for a role for the NBP in plant growth and development.

Non-NASA Center↗
NASA NTRS1993

A method to identify and characterize Z-DNA binding proteins using a linear oligodeoxynucleotide

An oligodeoxynucleotide that readily flips to the Z-DNA conformation in 10mM MgCl2 was produced by using Klenow enzyme to incorporate 5-bromodeoxycytosine and deoxyguanosine into a (dC-dG)22 template. During synthesis the oligomer can be labeled with 32P to high specific activity. The labeled oligodeoxynucleotide can be used in bandshift experiment to detect proteins that bind Z-DNA. This allows the binding specificity of such proteins to be determined with high reliability using unlabeled linear and supercoiled DNA competitors. In addition, because the radioactive oligodeoxynucleotide contains bromine atoms, DNA-protein complexes can be readily crosslinked using UV light. This allows an estimate to be made of the molecular weight of the proteins that bind to the radioactive probe. Both techniques are demonstrated using a goat polyclonal anti-Z-DNA antiserum.

NASA Program Exobiology↗
DOE OSTI2021

Calcium-Ion Binding Mediates the Reversible Interconversion of Cis and Trans Peroxido Dicopper Cores

Coupled dinuclear copper oxygen cores (Cu 2 O 2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O 2 transport and substrate oxidation in many organisms. μ-1,2-cis peroxido dicopper cores ( C P) have been proposed as key structures in the early stages of O 2 binding in these proteins; their reversible isomerization to other Cu 2 O 2 cores are directly relevant to enzyme function. Furthermore, despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems, the properties and reactivity of C P Cu 2 O 2 species remain largely unexplored. Herein, we report the reversible interconversion of μ-1,2-trans peroxido ( T P) and C P dicopper cores. Ca II mediates this process by reversible binding at the Cu 2 O 2 core, highlighting the unique capability for metal-ion binding events to stabilize novel reactive fragments and control O 2 activation in biomimetic systems.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Calcium‐Ion Binding Mediates the Reversible Interconversion of Cis and Trans Peroxido Dicopper Cores

Abstract Coupled dinuclear copper oxygen cores (Cu 2 O 2 ) featured in type III copper proteins (hemocyanin, tyrosinase, catechol oxidase) are vital for O 2 transport and substrate oxidation in many organisms. μ ‐1,2‐ cis peroxido dicopper cores ( C P ) have been proposed as key structures in the early stages of O 2 binding in these proteins; their reversible isomerization to other Cu 2 O 2 cores are directly relevant to enzyme function. Despite the relevance of such species to type III copper proteins and the broader interest in the properties and reactivity of bimetallic C P cores in biological and synthetic systems, the properties and reactivity of C P Cu 2 O 2 species remain largely unexplored. Herein, we report the reversible interconversion of μ ‐1,2‐ trans peroxido ( T P ) and C P dicopper cores. Ca II mediates this process by reversible binding at the Cu 2 O 2 core, highlighting the unique capability for metal‐ion binding events to stabilize novel reactive fragments and control O 2 activation in biomimetic systems.

Vargo, Natasha P.↗
DOE OSTI2023

Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS‐CoV‐2

Abstract Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‐CoV‐2) enters the host cell by binding to angiotensin‐converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS‐CoV‐2 viruses impact species specificity. Reverse zoonoses led to SARS‐CoV‐2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink‐associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio‐layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink‐derived S glycoproteins of SARS‐CoV‐2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo‐electron microscopy (cryo‐EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor‐binding domain (RBD) of the S glycoprotein in a “down” position, approximately 34° lower than previously reported “up” RBD. Finally, we compared residue interactions in the S‐ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS‐CoV‐2 entry.

Virology↗