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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Block Polyelectrolyte Additives That Modulate the Viscoelasticity and Enhance the Printability of Gelatin Inks at Physiological Temperatures

We demonstrate the utility of block polyelectrolyte (bPE) additives to enhance viscosity and resolve challenges with the three-dimensional (3D) printability of extrusion-based biopolymer inks. The addition of oppositely charged bPEs to solutions of photocurable gelatin methacryloyl (GelMA) results in complexation-driven self-assembly of the bPEs, leading to GelMA/bPE inks that are printable at physiological temperatures, representing stark improvements over GelMA inks that suffer from low viscosity at 37 °C, leading to low printability and poor structural stability. The hierarchical microstructure of the self-assemblies (either jammed micelles or 3D networks) formed by the oppositely charged bPEs, confirmed by small-angle X-ray scattering, is attributed to the enhancements in the shear strength and printability of the GelMA/bPE inks. Varying bPE concentration in the inks is shown to enable tunability of the rheological properties to meet the criteria of pre- and postextrusion flow characteristics for 3D printing, including prominent yielding behavior, strong shear thinning, and rapid recovery upon flow cessation. Moreover, the bPE self-assemblies also contribute to the robustness of the photo-cross-linked hydrogels; photo-cross-linked GelMA/bPE hydrogels are shown to exhibit higher shear strength than photo-cross-linked GelMA hydrogels. Last, the assessment of the printability of GelMA/bPE inks indicates excellent printing performance, including minimal swelling postextrusion, satisfactory retention of the filament shape upon deposition, and satisfactory shape fidelity of the various printed constructs. We envision this study to serve as a practical guide for the printing of bespoke extrusion inks where bPEs are used as scaffolds and viscosity enhancers that can be emulated in a range of photocurable precursors.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Instrumentation and experimental procedures for robust collection of X-ray diffraction data from protein crystals across physiological temperatures

Traditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure–function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Furthermore, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that can be directly related to biological function. Given this capability, the next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperature. This challenge is addressed herein. The approach described provides complete diffraction data sets with total collection times as short as ~5 s from single protein crystals, dramatically increasing the quantity of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09–1.54 Å resolution data over a temperature range of 293–363 K for proteinase K, thaumatin and lysozyme crystals at BL14-1 at the Stanford Synchrotron Radiation Lightsource. Here, the analyses presented here indicate that the diffraction data are of high quality and do not suffer from excessive dehydration or radiation damage.

36 MATERIALS SCIENCE↗
DOE OSTI2021

Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography

The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Dependence of thick filament structure in relaxed mammalian skeletal muscle on temperature and interfilament spacing

Contraction of skeletal muscle is regulated by structural changes in both actin-containing thin filaments and myosin-containing thick filaments, but myosin-based regulation is unlikely to be preserved after thick filament isolation, and its structural basis remains poorly characterized. Here, we describe the periodic features of the thick filament structure in situ by high-resolution small-angle x-ray diffraction and interference. We used both relaxed demembranated fibers and resting intact muscle preparations to assess whether thick filament regulation is preserved in demembranated fibers, which have been widely used for previous studies. We show that the thick filaments in both preparations exhibit two closely spaced axial periodicities, 43.1 nm and 45.5 nm, at near-physiological temperature. The shorter periodicity matches that of the myosin helix, and x-ray interference between the two arrays of myosin in the bipolar filament shows that all zones of the filament follow this periodicity. The 45.5-nm repeat has no helical component and originates from myosin layers closer to the filament midpoint associated with the titin super-repeat in that region. Cooling relaxed or resting muscle, which partially mimics the effects of calcium activation on thick filament structure, disrupts the helical order of the myosin motors, and they move out from the filament backbone. Compression of the filament lattice of demembranated fibers by 5% Dextran, which restores interfilament spacing to that in intact muscle, stabilizes the higher-temperature structure. The axial periodicity of the filament backbone increases on cooling, but in lattice-compressed fibers the periodicity of the myosin heads does not follow the extension of the backbone. Thick filament structure in lattice-compressed demembranated fibers at near-physiological temperature is similar to that in intact resting muscle, suggesting that the native structure of the thick filament is largely preserved after demembranation in these conditions, although not in the conditions used for most previous studies with this preparation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The impact of physiologically relevant temperatures on physical properties of thylakoid membranes: a molecular dynamics study

Thylakoid membranes are energy-converting membranes with a unique lipid composition. Though the membranes are primarily composed of proteins, their photosynthetic function is strongly influenced by the lipid constituents. Here we characterize, with molecular dynamics (MD) simulations, lipid bilayers with compositions representative of plant thylakoid membranes. We determine, in a wide range of temperatures, the physical parameters of the model membranes which are relevant for the photosynthetic function. We found a marked impact of temperature on membrane permeability due to a combination of increased compressibility and curvature of the membrane at elevated temperatures. With increasing temperatures, we observed increasingly smeared transmembrane density profiles of the membrane forming lipid headgroups predicting increased membrane flexibility. The diffusion coefficient of the lipids increased with temperature without apparent specificity for lipid species. Instead of a comprehensive experimental dataset in the relevant temperature range, we quantitatively compared and validated our MD results with MD simulations on a dipalmitoylphosphatidylcholine model system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Techniques to characterize dynamics in biomaterials microenvironments: XPCS and microrheology of alginate/PEO–PPO–PEO hydrogels

Many recent studies have highlighted the timescale for stress relaxation of biomaterials on the microscale as an important factor in regulating a number of cell-material interactions, including cell spreading, proliferation, and differentiation. Relevant timescales on the order of 0.1–100 s have been suggested by several studies. While such timescales are accessible through conventional mechanical rheology, several biomaterials have heterogeneous structures, and stress relaxation mechanisms of the bulk material may not correspond to that experienced in the cellular microenvironment. Here we employ X-ray photon correlation spectroscopy (XPCS) to explore the temperature-dependent dynamics, relaxation time, and microrheology of multicomponent hydrogels comprising of commercial poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO) triblock copolymer F127 and alginate. Previous studies on this system have shown thermoreversible behavior in the bulk oscillatory shear rheology. At physiological temperatures, bulk rheology of these samples shows behavior characteristic of a soft solid, with G' > G" and no crossover between G' and G" over the measurable frequency range, indicating a relaxation time >125 s. By contrast, XPCS-based microrheology shows viscoelastic behavior at low frequencies, and XPCS-derived correlation functions show relaxation times ranging from 10–45 s on smaller length scales. Finally, we are able to use XPCS to effectively probe the viscoelasticity and relaxation behavior within the material microenvironments.

36 MATERIALS SCIENCE↗
DOE OSTI2022

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to highthroughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required.

36 MATERIALS SCIENCE↗
DOE OSTI2020

Unusual zwitterionic catalytic site of SARS–CoV-2 main protease revealed by neutron crystallography

The main protease (3CL M pro ) from SARS–CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication. 3CL M pro possesses an unusual catalytic dyad composed of Cys 145 and His 41 residues. A critical question in the field has been what the protonation states of the ionizable residues in the substrate-binding active-site cavity are; resolving this point would help understand the catalytic details of the enzyme and inform rational drug development against this pernicious virus. Here, we present the room-temperature neutron structure of 3CL M pro , which allowed direct determination of hydrogen atom positions and, hence, protonation states in the protease. We observe that the catalytic site natively adopts a zwitterionic reactive form in which Cys 145 is in the negatively charged thiolate state and His 41 is doubly protonated and positively charged, instead of the neutral unreactive state usually envisaged. The neutron structure also identified the protonation states, and thus electrical charges, of all other amino acid residues and revealed intricate hydrogen-bonding networks in the active-site cavity and at the dimer interface. The fine atomic details present in this structure were made possible by the unique scattering properties of the neutron, which is an ideal probe for locating hydrogen positions and experimentally determining protonation states at near-physiological temperature. Our observations provide critical information for structure-assisted and computational drug design, allowing precise tailoring of inhibitors to the enzyme's electrostatic environment.

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

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF‐Trap

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Nanosecond time-resolved infrared spectroscopy for the study of electron transfer in photosystem I

Abstract Microsecond time-resolved step-scan FTIR difference spectroscopy was used to study photosystem I (PSI) from Thermosynechococcus vestitus BP-1 ( T. vestitus, formerly known as T. elongatus ) at 77 K. In addition, photoaccumulated (P700 + –P700) FTIR difference spectra were obtained at both 77 and 293 K. The FTIR difference spectra are presented here for the first time. To extend upon these FTIR studies nanosecond time-resolved infrared difference spectroscopy was also used to study PSI from T. vestitus at 296 K. Nanosecond infrared spectroscopy has never been used to study PSI samples at physiological temperatures, and here it is shown that such an approach has great value as it allows a direct probe of electron transfer down both branches in PSI. In PSI at 296 K, the infrared flash-induced absorption changes indicate electron transfer down the B- and A-branches is characterized by time constants of 33 and 364 ns, respectively, in good agreement with visible spectroscopy studies. These time constants are associated with forward electron transfer from A 1 – to F X on the B- and A-branches, respectively. At several infrared wavelengths flash-induced absorption changes at 296 K recover in tens to hundreds of milliseconds. The dominant decay phase is characterized by a lifetime of 128 ms. These millisecond changes are assigned to radical pair recombination reactions, with the changes being associated primarily with P700 + rereduction. This conclusion follows from the observation that the millisecond infrared spectrum is very similar to the photoaccumulated (P700 + –P700) FTIR difference spectrum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

X-ray Absorption Spectroscopy of Dilute Metalloenzymes at X-ray Free-Electron Lasers in a Shot-by-Shot Mode

X-ray absorption spectroscopy (XAS) of 3d transition metals provides important electronic structure information for many fields. However, X-ray-induced radiation damage under physiological temperature has prevented using this method to study dilute aqueous systems, such as metalloenzymes, as the catalytic reaction proceeds. Here we present a new approach to enable operando XAS of dilute biological samples and demonstrate its feasibility with K-edge XAS spectra from the Mn cluster in photosystem II and the Fe–S centers in photosystem I. This approach combines highly efficient sample delivery strategies and a robust signal normalization method with high-transmission Bragg diffraction-based spectrometers at X-ray free-electron lasers (XFELs) in a damage-free, shot-by-shot mode. These photon-out spectrometers have been optimized for discriminating the metal Mn/Fe Kα fluorescence signals from the overwhelming scattering background present on currently available detectors for XFELs that lack suitable energy discrimination. We quantify the enhanced performance metrics of the spectrometer and discuss its potential applications for acquiring time-resolved XAS spectra of biological samples during their reactions at XFELs.

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

Olivine Dissolution in Simulated Lung and Gastric Fluid as an Analog to the Behavior of Lunar Particulate Matter Inside the Human Respiratory and Gastrointestinal Systems

With the Artemis III mission scheduled to land humans on the Moon in 2025, work must be done to understand the hazards lunar dust inhalation would pose to humans. In this study, San Carlos olivine was used as an analog of lunar olivine, a common component of lunar dust. Olivine was dissolved in a flow-through apparatus in both simulated lung fluid and 0.1 M HCl (simulated gastric fluid) over a period of approximately 2 weeks at physiological temperature, 37°C. Effluent samples were collected periodically and analyzed for pH, iron, silicon, and magnesium ion concentrations. The dissolution rate data derived from our measurements allow us to estimate that an inhaled 1.0 μm diameter olivine particle would take approximately 24 years to dissolve in the human lungs and approximately 3 weeks to dissolve in gastric fluid. Results revealed that inhaled olivine particles may generate the toxic chemical, hydroxyl radical, for up to 5–6 days in lung fluid. Olivine dissolved in 0.1 M HCl for 2 weeks transformed to an amorphous silica-rich solid plus the ferric iron oxy-hydroxide ferrihydrite. Olivine dissolved in simulated lung fluid shows no detectable change in composition or crystallinity. Equilibrium thermodynamic models indicate that olivine in the human lungs can precipitate secondary minerals with fibrous crystal structures that have the potential to induce detrimental health effects similar to asbestos exposure. Our work indicates that inhaled lunar dust containing olivine can settle in the human lungs for years and could induce long-term potential health effects like that of silicosis.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗
DOE OSTI2022

EWALD: A macromolecular diffractometer for the second target station

Revealing the positions of all the atoms in large macromolecules is powerful but only possible with neutron macromolecular crystallography (NMC). Neutrons provide a sensitive and gentle probe for the direct detection of protonation states at near-physiological temperatures and clean of artifacts caused by x rays or electrons. Currently, NMC use is restricted by the requirement for large crystal volumes even at state-of-the-art instruments such as the macromolecular neutron diffractometer at the Spallation Neutron Source. EWALD’s design will break the crystal volume barrier and, thus, open the door for new types of experiments, the study of grand challenge systems, and the more routine use of NMC in biology. EWALD is a single crystal diffractometer capable of collecting data from macromolecular crystals on orders of magnitude smaller than what is currently feasible. The construction of EWALD at the Second Target Station will cause a revolution in NMC by enabling key discoveries in the biological, biomedical, and bioenergy sciences.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2023

Molecular recognition requires dimerization of a VHH antibody

Camelid heavy-chain-only antibodies are a unique class of antibody that possesses only a single variable domain (termed VHH) for antigen recognition. Despite their apparent canonical mechanism of target recognition, where a single VHH domain binds a single target, an anti-caffeine VHH has been observed to possess 2:1 stoichiometry. Here, the structure of the anti-caffeine VHH/caffeine complex enabled the generation and biophysical analysis of variants that were used to better understand the role of VHH homodimerization in caffeine recognition. VHH interface mutants and caffeine analogs, which were examined to probe the mechanism of caffeine binding, suggested caffeine recognition is only possible with the VHH dimer species. Correspondingly, in the absence of caffeine, the anti-caffeine VHH was found to form a dimer with a dimerization constant comparable to that observed with VH:VL domains in conventional antibody systems, which was most stable near physiological temperature. While the VHH:VHH dimer structure (at 1.13 Å resolution) is reminiscent of conventional VH:VL heterodimers, the homodimeric VHH possesses a smaller angle of domain interaction, as well as a larger amount of apolar surface area burial. To test the general hypothesis that the short complementarity-determining region-3 (CDR3) may help drive VHH:VHH homodimerization, an anti-picloram VHH domain containing a short CDR3 was generated and characterized, which revealed it also existed as dimer species in solution. These results suggest homodimer-driven recognition may represent a more common method of VHH ligand recognition, opening opportunities for novel VHH homodimer affinity reagents and helping to guide their use in chemically induced dimerization applications.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Fixed-target serial crystallography at the Structural Biology Center

Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two β-lactamases: a class D serine β-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-β-lactamase from Stenotrophomonas maltophilia K279a.

59 BASIC BIOLOGICAL SCIENCES↗