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All sources NASA NTRS DOE OSTI

DOE OSTI2022

Substrate multiplexed protein engineering facilitates promiscuous biocatalytic synthesis

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

INBOUND APPOINTEE PUBLICATION Importance of Substrate-Particle Repulsion for Protein-Templated Assembly of Metal Nanoparticles

We study the protein-directed assembly of colloidal gold nanoparticles on de novo designed protein nanofiber templates. Using sequential assembly on glass substrates, we attach of positively-charged gold nanoparticles to protein nanofibers engineered to have a high density of negatively-charged surface residues. Using a combination of electron and optical microscopy, we measure the density of particle attachment and characterize binding specificity. By varying nanoparticle size and pH of the solution, we explore the importance of charge-dependent particle-fiber and particle-substrate interactions. We find an inverse correlation between particle size and attachment density to the protein nanofibers, attributed to the balance between size-dependent electrostatic particle-fiber attraction and particle-substrate repulsion. We show pH-dependent particle attachment density and binding specificity in relation to the protonation fraction of each assembly layer. Finally, we employ hyperspectral scattering microscopy to draw conclusions about particle density and interparticle spacings of optically-observable particle assemblies.

Guye, Kathryn N.↗

DOE OSTI2016

Structural and Functional Role of Acetyltransferase hMOF K274 Autoacetylation

Many histone acetyltransferases undergo autoacetylation, either through chemical or enzymatic means, to potentiate enzymatic cognate substrate lysine acetylation, although the mode and molecular role of such autoacetylation is poorly understood. The MYST family of histone acetyltransferases is autoacetylated at an active site lysine residue to facilitate cognate substrate lysine binding and acetylation. Here, we report on a detailed molecular investigation of Lys-274 autoacetylation of the human MYST protein Males Absent on the First (hMOF). A mutational scan of hMOF Lys-274 reveals that all amino acid substitutions of this residue are able to bind cofactor but are significantly destabilized, both in vitro and in cells, and are catalytically inactive for cognate histone H4 peptide lysine acetylation. The x-ray crystal structure of a hMOF K274P mutant suggests that the reduced stability and catalytic activity stems from a disordering of the residue 274-harboring a α2-β7 loop. We also provide structural evidence that a C316S/E350Q mutant, which is defective for cognate substrate lysine acetylation; and biochemical evidence that a K268M mutant, which is defective for Lys-274 chemical acetylation in the context of a K274-peptide, can still undergo quantitative K274 autoacetylation. Together, these studies point to the critical and specific role of hMOF Lys-274 autoacetylation in hMOF stability and cognate substrate acetylation and argues that binding of Ac-CoA to hMOF likely drives Lys-274 autoacetylation for subsequent cognate substrate acetylation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2015

Advanced Mirror Technology Development (AMTD) Project: 3.0 Year Status

Advanced Mirror Technology Development (AMTD) is a funded NASA Strategic Astrophysics Technology project. Begun in 2011, we are in Phase 2 of a multi-year effort. Our objective is to mature towards TRL6 critical technologies needed to produce 4-m or larger flight-qualified UVOIR mirrors by 2018 so that a viable astronomy mission can be considered by the 2020 Decadal Review. The developed technology must enable missions capable of both general astrophysics and ultra-high contrast observations of exoplanets. Just as JWST's architecture was driven by launch vehicle, a future UVOIR mission's architecture (monolithic, segmented or interferometric) will depend on capacities of future launch vehicles (and budget). Since we cannot predict the future, we must prepare for all potential futures. Therefore, we are pursuing multiple technology paths. AMTD uses a science-driven systems engineering approach. We mature technologies required to enable the highest priority science AND result in a high-performance low-cost low-risk system. One of our key accomplishments is that we have derived engineering specifications for advanced normal-incidence monolithic and segmented mirror systems needed to enable both general astrophysics and ultra-high contrast observations of exoplanets missions as a function of potential launch vehicle and its inherent mass and volume constraints. Another key accomplishment is that we have matured our technology by building and testing hardware. To demonstrate stacked core technology, we built a 400 mm thick mirror. Currently, to demonstrate lateral scalability, we are manufacturing a 1.5 meter mirror. To assist in architecture trade studies, the Engineering team develops Structural, Thermal and Optical Performance (STOP) models of candidate mirror assembly systems including substrates, structures, and mechanisms. These models are validated by test of full- and subscale components in relevant thermo-vacuum environments. Specific analyses include: maximum mirror substrate size, first fundamental mode frequency (i.e., stiffness) and mass required to fabricate without quilting, survive launch, and achieve stable pointing and maximum thermal time constant.

Stahl, H. Philip↗

DOE OSTI2016

Effect of the nature of the substrate on the surface chemistry of atomic layer deposition precursors

The thermal chemistry of Cu(I)-sec-butyl-2-iminopyrrolidinate, a promising copper amidinate complex for atomic layer deposition (ALD) applications, was explored comparatively on several surfaces by using a combination of surface-sensitive techniques, specifically temperature-programmed desorption and x-ray photoelectron spectroscopy (XPS). The substrates explored include single crystals of transition metals (Ni(110) and Cu(110)), thin oxide films (NiO/Ni(110) and SiO 2 /Ta), and oxygen-treated metals (O/Cu(110)). Decomposition of the pyrrolidinate ligand leads to the desorption of several gas-phase products, including CH 3 CN, HCN and butene from the metals and CO and CO 2 from the oxygen-containing surfaces. In all cases dehydrogenation of the organic moieties is accompanied by hydrogen removal from the surface, in the form of H 2 on metals and mainly as water from the metal oxides, but the threshold for this chemistry varies wildly, from 270 K on Ni(110) to 430 K on O/Cu(110), 470 K on Cu(110), 500 K on NiO/Ni(110), and 570 K on SiO 2 /Ta. Here, copper reduction is also observed in both the Cu 2p 3/2 XPS and the Cu L 3 VV Auger (AES) spectra, reaching completion by 300 K on Ni(110) but occurring only between 500 and 600 K on Cu(110). On NiO/Ni(110), both Cu(I) and Cu(0) coexist between 200 and 500 K, and on SiO 2 /Ta a change happens between 500 and 600 K but the reduction is limited, with the copper atoms retaining a significant ionic character. Additional experiments to test adsorption at higher temperatures led to the identification of temperature windows for the self-limiting precursor uptake required for ALD between approximately 300 and 450 K on both Ni(110) and NiO/Ni(110); the range on SiO 2 had been previously determined to be wider, reaching an upper limit at about 500 K. Finally, deposition of copper metal films via ALD cycles with O 2 as the co-reactant was successfully accomplished on the Ni(110) substrate.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2014

Methods and systems for Raman and optical cross-interrogation in flow-through silicon membranes

Cross-interrogating photonic detection systems and methods are shown. A flow through photonic crystal membrane with a surface enhanced Raman scattering (SERS) substrate is provided with pores which are distributed along multiple regions. The pores of one region have walls to which a first type of target specific anchor can be attached, while pores of another region have walls to which a second type of target specific anchor can be attached. An optical arrangement out-of-plane to the SERS substrate is also provided for enhanced sensitivity and identification of target organisms.

47 OTHER INSTRUMENTATION↗

DOE OSTI2015

Electron microscopy of an aluminum layer grown on the vicinal surface of a gallium arsenide substrate

A thin Al layer grown by molecular-beam epitaxy on a misoriented GaAs (100) substrate is studied by transmission electron microscopy. Electron diffraction data and bright-field, dark-field, and high-resolution images show that, in the layer, there are Al grains of three types of crystallographic orientation: Al (100), Al (110), and Al (110)R. The specific structural features of the interfaces between the differently oriented grains and substrate are studied by digital processing of the high-resolution images. From quantitative analysis of the dark-field images, the relative content and sizes of the differently oriented grains are determined. It is found that atomic steps at the substrate surface cause an increase in the fraction and sizes of Al (110)R grains and a decrease in the fraction of Al (100) grains, compared to the corresponding fractions and sizes in the layer grown on a singular substrate surface.

36 MATERIALS SCIENCE↗

DOE OSTI2020

CRISPR-Cas “Non-Target” Sites Inhibit On-Target Cutting Rates

CRISPR-Cas systems have become ubiquitous for genome editing in eukaryotic as well as bacterial systems. Cas9 forms a complex with a guide RNA (gRNA) and searches DNA for a matching sequence (target site) next to a protospacer adjacent motif (PAM). Once found, Cas9 cuts the DNA. Cas9 is revolutionary for the ability to change the RNA sequence and target a new site easily. However, while algorithms have been developed to predict gRNA-specific Cas9 activity, a fundamental biological understanding of gRNA-specific activity is lacking. Here, the number of PAM sites in the genome is effectively a large pool of inhibitory substrates, competing with the target site for the Cas9/gRNA complex. We demonstrate that increasing the number of non-target sites for a given gRNA reduces on-target activity in a dose-dependent manner. Furthermore, we show that the use of Cas9 mutants with increased PAM specificity toward a smaller subset of PAMs (or smaller pool of competitive substrates) improves cutting rates, while increased PAM promiscuity decreases cutting rates. Decreasing the potential search space by increasing PAM specificity provides a path toward improving on-target activity for slower high-fidelity Cas9 variants. Engineering improved PAM specificity to reduce the competitive search space offers an alternative strategy to engineer Cas9 variants with increased specificity and maintained on-target activity.

24 POWER TRANSMISSION AND DISTRIBUTION↗

DOE OSTI2024

Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi

Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Optical and photoelectric properties of surface barrier diodes based on n- Hg3In2Te6 crystals

The high radiation resistance of Hg3In2Te6 (MIT) single crystals to large doses of ionizing radiation makes devices quite attractive. Such a feature of the crystal structure has not only important advantages, but also causes serious technological difficulties in doping and controlling the specific resistance within the required limits and type of conductivity of single crystals. These technological difficulties create serious problems when creating structures with a potential barrier, especially with a p-n junction. At the moment, there are no publications on the creation of a p-n junction based on MIT. The only alternative is to create a potential barrier such as a Schottky diode or a metal-dielectric-semiconductor structure. This limits the use of MIT to produce, for example, rectifier diodes that can operate at relatively high reverse bias voltages. The typical resistivity of single crystals of MIT of n-type conductivity, which are used for the manufacture of diodes and photodiodes, is approximately equal to ρ≈103-104 Ω×cm. Such a specific resistance leads to a relatively large resistance of the neutral part of the single crystal, which in turn limits the direct currents of the rectifier diodes and significantly limits an important parameter of photodiodes - the dynamic range of the watt-ampere characteristic. This problem arises from the relatively high series resistance of the substrate. For rectifier diodes, this relatively large series resistance leads to a significant forward bias voltage drop, which reduces both the rectification factor and the amount of rectified current. We have already created diodes and photodiodes on Cr/MIT/Cr single crystals with a specific resistance of ρ≈103-104 Ω×cm. The diodes had relatively small dark currents but had the disadvantage of a relatively large specific resistance of the substrate, which we have already discussed above [1,2,3].

Sklyarchuk, V.↗

DOE OSTI2020

A Low-Cost Iron-Based Current Collector for Alkaline Battery Electrodes

The use of three-dimensional porous nickel foam as the current collector of the nickel hydroxide electrode adds significantly to the cost of the nickel-based alkaline rechargeable batteries. Although iron is considerably less expensive than nickel, iron corrodes at the operating potential of the nickel hydroxide electrode. We have found that a 70–100 nm thick thermal coating of cobalt ferrite spinel protects the iron from corrosion. Such a coated iron substrate was found to be stable against corrosion even when polarized anodically at 10 mA cm -2 in 30% potassium hydroxide electrolyte for 1000 h. While the thermal coating of cobalt ferrite protected iron against corrosion, incorporation of lithium ions into the coating was found to enhance the electrical conductivity of the coating. XPS and EXAFS studies confirmed that the enhanced conductivity resulted from an increase in the population of Co 3+ in the ferrite spinel lattice. An inexpensive iron (steel) substrate protected by such a coating when used as a nickel hydroxide battery electrode exhibited a specific capacity of 0.25 Ah g -1 at C/5 discharge rate, comparable to a nickel hydroxide electrode based on a relatively expensive nickel foam substrate. The steel-based electrode also exhibited no noticeable degradation over 150 cycles at C/2 rate. This demonstration of a robust and economical steel substrate presents a unique opportunity for reducing the cost of the nickel hydroxide battery electrode in alkaline batteries

25 ENERGY STORAGE↗

DOE OSTI2015

The Majorana low-noise low-background front-end electronics

The Majorana Demonstrator will search for the neutrinoless double beta decay (ββ(0ν)) of the isotope ⁷⁶Ge with a mixed array of enriched and natural germanium detectors. In view of the next generation of tonne-scale germanium-based ββ(0ν)-decay searches, a major goal of the Majorana Demonstrator is to demonstrate a path forward to achieving a background rate at or below 1 cnt/(ROI-t-y) in the 4 keV region of interest (ROI) around the 2039-keV Q-value of the ⁷⁶Ge ββ(0ν)-decay. Such a requirement on the background level significantly constrains the design of the readout electronics, which is further driven by noise and energy resolution performances. We present here the low-noise low-background front-end electronics developed for the low-capacitance p-type point contact (P-PC) germanium detectors of the Majorana Demonstrator. This resistive-feedback front-end, specifically designed to have low mass, is fabricated on a radioassayed fused-silica substrate where the feedback resistor consists of a sputtered thin film of high purity amorphous germanium and the feedback capacitor is based on the capacitance between gold conductive traces.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2022

Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP 8 . IP 8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP 8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg 2+ or Mn 2+ ; IP 6 , 5-IP 7 , or 1,5-IP 8 ) and the other which was ligand-free. The ligand-free enzyme adopts an “open” conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP 6 or 5-IP 7 engagement above the nucleotide favors adoption of a “closed” conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP 7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N 6 . Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N 6 -benzyl-ATP as phosphate donor.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

A minimal motif for sequence recognition by mitochondrial transcription factor A (TFAM)

Abstract Mitochondrial transcription factor A (TFAM) plays a critical role in mitochondrial transcription initiation and mitochondrial DNA (mtDNA) packaging. Both functions require DNA binding, but in one case TFAM must recognize a specific promoter sequence, while packaging requires coating of mtDNA by association with non sequence-specific regions. The mechanisms by which TFAM achieves both sequence-specific and non sequence-specific recognition have not yet been determined. Existing crystal structures of TFAM bound to DNA allowed us to identify two guanine-specific interactions that are established between TFAM and the bound DNA. These interactions are observed when TFAM is bound to both specific promoter sequences and non-sequence specific DNA. These interactions are established with two guanine bases separated by 10 random nucleotides (GN10G). Our biochemical results demonstrate that the GN10G consensus is essential for transcriptional initiation and contributes to facilitating TFAM binding to DNA substrates. Furthermore, we report a crystal structure of TFAM in complex with a non sequence-specific sequence containing a GN10G consensus. The structure reveals a unique arrangement in which TFAM bridges two DNA substrates while maintaining the GN10G interactions. We propose that the GN10G consensus is key to facilitate the interaction of TFAM with DNA.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Phonon spectra of a two-dimensional solid dusty plasma modified by two-dimensional periodic substrates

Here, phonon spectra of a two-dimensional (2D) solid dusty plasma modulated by 2D square and triangular periodic substrates are investigated using Langevin dynamical simulations. The commensurability ratio, i.e., the ratio of the number of particles to the number of potential well minima, is set to 1 or 2. The resulting phonon spectra show that propagation of waves is always suppressed due to the confinement of particles by the applied 2D periodic substrates. For a commensurability ratio of 1, the spectra indicate that all particles mainly oscillate at one specific frequency, corresponding to the harmonic oscillation frequency of one single particle inside one potential well. At a commensurability ratio of 2, the substrate allows two particles to sit inside the bottom of each potential well, and the resulting longitudinal and transverse spectra exhibit four branches in total. We find that the two moderate branches come from the harmonic oscillations of one single particle and two combined particles in the potential well. The other two branches correspond to the relative motion of the two-body structure in each potential well in the radial and azimuthal directions. The difference in the spectra between the square and triangular substrates is attributed to the anisotropy of the substrates and the resulting alignment directions of the two-body structure in each potential well.

36 MATERIALS SCIENCE↗

NASA NTRS2002

Single Source Precursors for Thin Film Solar Cells

The development of thin film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. At NASA GRC we have focused on the development of new single source precursors (SSP) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD (chemical vapor deposition) process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV (photovoltaic) devices.

Banger, Kulbinder K.↗

DOE OSTI2015

Order parameters and magnetocrystalline anisotropy of off-stoichiometric D0{sub 22} Mn{sub 2.36}Ga epitaxial films grown on MgO (001) and SrTiO{sub 3} (001)

We study the relationship between long range order parameters and the magnetocrystalline anisotropy of off-stoichiometric D0{sub 22} Mn{sub 2.36}Ga (MnGa) epitaxial films grown on MgO (001) and SrTiO{sub 3} (STO) (001) single crystalline substrates. MnGa films deposited on MgO (001) show rather large irregular variation in magnetization with increasing substrate temperature in spite of the improved long range order of total atomic sites. The specific site long range order of Mn-I site characterized in the [101] orientation revealed the fluctuation of the occupation fraction of two Mn atomic sites with elevated substrate temperature, which appears more relevant to the observed magnetization change than the long range order of the total atomic sites. In case of MnGa films grown on the lattice-matched STO (001), high long range order of the total atomic sites in spite of the existence of secondary phase represents that the lattice mismatch plays a crucial role in determining the atomic arrangement of Mn and Ga atoms in the off-stoichiometric compositional case of MnGa.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

DOE OSTI2015

Nanoscale carbon materials from hydrocarbons pyrolysis: Structure, chemical behavior, utilisation for non-aqueous supercapacitors

Highlights: • N-doped and regular carbon nanomaterials were obtained by pyrolitic technique. • Dynamic vapor sorption of different solvents reveals smaller S{sub BET} values. • Steric hindrance and specific chemical interactions are the reasons for this. • Nitrogen doping leads to raise of capacitance and coulombic efficiency with non-aqueous N-containing electrolyte. - Abstract: This work systematically studies adsorption properties of carbon nanomaterials that are synthesized through hydrocarbons that is a powerful technique to fabricate different kinds of carbon materials, e.g., nanotubes, nanoshells, onions, including nitrogen substituted. The adsorption properties of the as-synthesized carbons are achieved by low temperature nitrogen adsorption and organic vapors sorption. Heptane, acetonitrile, water, ethanol, benzene and 1-methylimidazole, which are of great importance for development of supercapacitors, are used as substrates. It is discovered that while nitrogen adsorption reveals a high specific surface area, this parameter for most of organic compounds is rather small depending not only on the size of its molecule but also on chemical interactions for a pair adsorbent–adsorbate. The experimental values of heat of adsorption for carbon and N-substituted structures, when Coulomb cross-coupling of nitrogen atoms in adsorbent and adsorbate takes place, confirms this supposition.

77 NANOSCIENCE AND NANOTECHNOLOGY↗