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DOE OSTI2019

Vapor-Deposited Glass Structure Determined by Deposition Rate–Substrate Temperature Superposition Principle

In this article, we show that deposition rate substantially affects the anisotropic structure of thin glassy films produced by physical vapor deposition. Itraconazole, a glass-forming liquid crystal, was deposited at rates spanning 3 orders of magnitude over a 25 K range of substrate temperatures, and structure was characterized by ellipsometry and X-ray scattering. Both the molecular orientation and the spacing of the smectic layers obey deposition rate–substrate temperature superposition, such that lowering the deposition rate is equivalent to raising the substrate temperature. We identify two different surface relaxations that are responsible for structural order in the vapor-deposited glasses and find that the process controlling molecular orientation is accelerated by more than 3 orders of magnitude at the surface relative to the bulk. Finally, the identification of distinct surface processes responsible for anisotropic structural features in vapor-deposited glasses will enable more precise control over the structure of glassy materials used in organic electronics.

36 MATERIALS SCIENCE↗

DOE OSTI2025

Unraveling Enhanced Superconductivity in Single-Layer FeSe through Substrate Surface Terminations

Single-layer FeSe on SrTiO 3 (001) substrates shows a superconducting transition temperature much higher than that of bulk FeSe, which has been attributed to factors such as electron doping, interfacial electron–phonon coupling, and electron correlations. To pinpoint the primary driver, we grew single-layer FeSe films on SrTiO 3 (001) substrates with coexisting TiO 2 and SrO surface terminations. Scanning tunneling spectroscopy revealed a larger superconducting gap (17.0 meV) on the TiO 2 -termination than on the SrO-termination (10.5 meV). Tunneling spectroscopy also showed a larger work function on the SrO surface, resulting in reduced charge transfer to FeSe, as confirmed by angle-resolved photoemission spectroscopy. Scanning transmission electron microscopy further revealed distinctive interfacial atomic-scale structures, with the Se–Fe–Se tetrahedral angle changing from 109.5° on the SrO-termination to 104.9° on the TiO 2 -termination. Compared to dynamical mean field theory calculations, our results indicate that enhanced superconductivity in single-layer FeSe/TiO 2 arises from optimal electron correlations, in addition to sufficient charge transfer from the substrate.

75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND↗

DOE OSTI2018

Substrate Binding Induces Conformational Changes in a Class A β-lactamase That Prime It for Catalysis

Here, the emergence and dissemination of bacterial resistance to β-lactam antibiotics via β-lactamase enzymes is a serious problem in clinical settings, often leaving few treatment options for infections resulting from multidrug-resistant superbugs. Understanding the catalytic mechanism of β-lactamases is important for developing strategies to overcome resistance. Binding of a substrate in the active site of an enzyme can alter the conformations and p K a s of catalytic residues, thereby contributing to enzyme catalysis. Here we report X-ray and neutron crystal structures of the class A Toho-1 β-lactamase in the apo form and an X-ray structure of a Michaelis-like complex with the cephalosporin antibiotic cefotaxime in the active site. Comparison of these structures reveals that substrate binding induces a series of changes. The side chains of conserved residues important in catalysis, Lys73 and Tyr105, and the main chain of Ser130 alter their conformations, with Nζ of Lys73 moving closer to the position of the conserved catalytic nucleophile Ser70. This movement of Lys73 closer to Ser70 is consistent with proton transfer between the two residues prior to acylation. In combination with the tightly bound catalytic water molecule located between Glu166 and the position of Ser70, the enzyme is primed for catalysis when Ser70 is activated for nucleophilic attack of the β-lactam ring. Quantum mechanical/molecular mechanical (QM/MM) free energy simulations of models of the wild-type enzyme show that proton transfer from the Nζ of Lys73 to the Oε2 atom of Glu166 is more thermodynamically favorable than when it is absent. Taken together, our findings indicate that substrate binding enhances the favorability of the initial proton transfer steps that precede the formation of the acyl-enzyme intermediate.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Transfer-Free Synthesis of Atomically Precise Graphene Nanoribbons on Insulating Substrates

The rational bottom-up synthesis of graphene nanoribbons (GNRs) provides atomically precise control of widths and edges that give rise to a wide range of electronic properties promising for electronic devices such as field-effect transistors (FETs). Since the bottom-up synthesis commonly takes place on catalytic metallic surfaces, the integration of GNRs into such devices requires their transfer onto insulating substrates, which remains one of the bottlenecks in the development of GNR-based electronics. Herein, we report on a method for the transfer-free placement of GNRs on insulators. This involves growing GNRs on a gold film deposited onto an insulating layer followed by gentle wet etching of the gold, which leaves the nanoribbons to settle in place on the underlying insulating substrate. Scanning tunneling microscopy and Raman spectroscopy confirm that atomically precise GNRs of high density uniformly grow on the gold films deposited onto SiO 2 /Si substrates and remain structurally intact after the etching process. We have also demonstrated transfer-free fabrication of ultrashort channel GNR FETs using this process. A very important aspect of the present work is that the method can scale up well to 12 in. wafers, which is extremely difficult for previous techniques. Our work here thus represents an important step toward large-scale integration of GNRs into electronic devices.

36 MATERIALS SCIENCE↗

DOE OSTI2026

Substrate-Directed Underlayer Growth of Bilayer MoS 2 Revealed by Mo Isotope Labeling

Direct control over the vertical formation sequence and stacking registry in van der Waals (vdW) bilayers is essential for device performance and moiré engineering yet difficult to resolve unambiguously with conventional probes. Here, we use Mo isotope labeling in a two-step chemical vapor deposition process to synthesize bilayer MoS 2 and trace its vertical formation on common substrates. By combining site-selective laser thinning, Raman spectroscopy, time-of-flight secondary ion mass spectrometry, and atomic-resolution scanning transimission electron microscopy (STEM), we find a clear substrate dependence: on SiO 2 /Si, the second layer nucleates and grows beneath the first (underlayer), whereas on sapphire, it forms on top (overlayer). Density functional theory indicates that a larger equilibrium interfacial separation and weaker MoS 2 –substrate interactions on amorphous SiO 2 permit confined interfacial diffusion and underlayer nucleation, whereas stronger interactions and smaller separations on sapphire favor overlayer growth. On SiO 2 , confined epitaxy templates commensurate 2H, 3R, and mixed bilayers, as confirmed by second harmonic generation spectroscopy and STEM. During underlayer coalescence, embedded mirror-twin grain boundaries stitch atomically sharp 2H|3R junctions via alternating 4|8 ring motifs. Molecular-dynamics simulations reveal that these alternating 4|8 motifs accommodate interlayer vdW coupling and locally modulate the stacking registry. These results provide mechanistic insight into confined epitaxial growth and establish isotope labeling as a powerful probe of two-dimensional materials synthesis.

MoS2↗

DOE OSTI2026

Production and Evaluation of Fluorophore-Doped Polymer Substrates to Screen for Plastic-Degrading Enzymes

Fast and sensitive analytical methods are the key to efficient screening of plastic-degrading enzymes. Here, we present a streamlined and affordable approach to assess the enzymatic deconstruction of insoluble synthetic polymers by blending them with a fluorescent dye, rhodamine 6G, and we evaluate this screening method using poly(ethylene terephthalate) (PET) as a model material. Our results indicate that enzymatic depolymerization of the rhodamine-doped PET can be observed in a high-throughput fashion by following release of the fluorophore. The fluorescence data obtained during the hydrolysis of rhodamine-doped PET by 14 PET hydrolases, produced with a robotic platform, correlated with the quantitative chromatographic analysis of PET degradation products. Remarkably, the use of the rhodamine-loaded PET substrate resulted in negligibly low background signals even when detecting PETase activity in crude cell lysates, suggesting suitability for screening of a wide variety of samples. Encouraged by these results, we next produced a selection of polyethylene- and nylon-based materials loaded with rhodamine 6G. While rapid leaching of fluorophore observed with nylon substrates limits the utility of the method for detecting nylonase activity, the rhodamine-loaded polyethylene showed promising performance in passive diffusion tests, indicating that this latter substrate may be used to screen for polyolefin-degrading enzymes.

09 BIOMASS FUELS↗

DOE OSTI2020

Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment

Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination of an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

Lithium-ion electrolytic substrates for sub-1V high-performance transition metal dichalcogenide transistors and amplifiers

Electrostatic gating of two-dimensional (2D) materials with ionic liquids (ILs), leading to the accumulation of high surface charge carrier densities, has been often exploited in 2D devices. However, the intrinsic liquid nature of ILs, their sensitivity to humidity, and the stress induced in frozen liquids inhibit ILs from constituting an ideal platform for electrostatic gating. Here we report a lithium-ion solid electrolyte substrate, demonstrating its application in high-performance back-gated n-type MoS 2 and p-type WSe 2 transistors with sub-threshold values approaching the ideal limit of 60 mV/dec and complementary inverter amplifier gain of 34, the highest among comparable amplifiers. Remarkably, these outstanding values were obtained under 1 V power supply. Microscopic studies of the transistor channel using microwave impedance microscopy reveal a homogeneous channel formation, indicative of a smooth interface between the TMD and underlying electrolytic substrate. These results establish lithium-ion substrates as a promising alternative to ILs for advanced thin-film devices.

42 ENGINEERING↗

DOE OSTI2022

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

Presequence protease (PreP), a 117kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3–4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Analysis of diverse double-strand break synapsis with Polλ reveals basis for unique substrate specificity in nonhomologous end-joining

DNA double-strand breaks (DSBs) threaten genomic stability, since their persistence can lead to loss of critical genetic information, chromosomal translocations or rearrangements, and cell death. DSBs can be repaired through the nonhomologous end-joining pathway (NHEJ), which processes and ligates DNA ends efficiently to prevent or minimize sequence loss. Polymerase λ (Polλ), one of the Family X polymerases, fills sequence gaps of DSB substrates with a strict specificity for a base-paired primer terminus. There is little information regarding Polλ’s approach to engaging such substrates. We used in vitro polymerization and cell-based NHEJ assays to explore the contributions of conserved loop regions toward DSB substrate specificity and utilization. In addition, we present multiple crystal structures of Polλ in synapsis with varying biologically relevant DSB end configurations, revealing how key structural features and hydrogen bonding networks work in concert to stabilize these tenuous, potentially cytotoxic DNA lesions during NHEJ.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Computational remodeling of an enzyme conformational landscape for altered substrate selectivity

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Substrate binding plasticity revealed by Cryo-EM structures of SLC26A2

SLC26A2 is a vital solute carrier responsible for transporting essential nutritional ions, including sulfate, within the human body. Pathogenic mutations within SLC26A2 give rise to a spectrum of human diseases, ranging from lethal to mild symptoms. The molecular details regarding the versatile substrate-transporter interactions and the impact of pathogenic mutations on SLC26A2 transporter function remain unclear. Here, using cryo-electron microscopy, we determine three high-resolution structures of SLC26A2 in complexes with different substrates. These structures unveil valuable insights, including the distinct features of the homodimer assembly, the dynamic nature of substrate binding, and the potential ramifications of pathogenic mutations. This structural-functional information regarding SLC26A2 will advance our understanding of cellular sulfate transport mechanisms and provide foundations for future therapeutic development against various human diseases.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

The molecular basis of Human FN3K mediated phosphorylation of glycated substrates

Abstract Glycation, a non-enzymatic post-translational modification occurring on proteins, can be actively reversed via site-specific phosphorylation of the fructose-lysine moiety by FN3K kinase, to impact the cellular function of the target protein. A regulatory axis between FN3K and glycated protein targets has been associated with conditions like diabetes and cancer. However, the molecular basis of this relationship has not been explored so far. Here, we determined a series of crystal structures of HsFN3K in the apo-state, and in complex with different nucleotide analogs together with a sugar substrate mimic to reveal the features important for its kinase activity and substrate recognition. Additionally, the dynamics in sugar substrate binding during the kinase catalytic cycle provide important mechanistic insights into HsFN3K function. Our structural work provides the molecular basis for rational small molecule design targeting FN3K.

Science & Technology - Other Topics↗

DOE OSTI2025

A substrate-multiplexed platform for profiling enzymatic potential of plant family 1 glycosyltransferases

Plants have expanded various biosynthetic enzyme families to produce a wide diversity of natural products; however, most enzymes encoded in plant genomes remain uncharacterized, highlighting the need for new functional genomic approaches. Here, we report a platform enabling the rapid functional characterization of plant family 1 glycosyltransferases, which serve important roles in plant development, defense, and communication. Using substrate-multiplexed reactions, mass spectrometry, and automated analysis, we screen 85 enzymes against a diverse library of 453 natural products, for a total of nearly 40,000 possible reactions. The resulting dataset reveals a widespread promiscuity and a strong preference for planar, hydroxylated aromatic substrates among family 1 glycosyltransferases. We also characterize glycosyltransferases with an unusually wide substrate scope and with a non-canonical Cys-Asp catalytic dyad. This work establishes a widely-applicable enzymatic screening pipeline, reflects the immense glycosylation capability of plants, and has implications in biocatalysis, metabolic engineering, and gene discovery.

Sirirungruang, Sasilada↗

DOE OSTI2021

Thermal stability and diffusion characteristics of ultrathin amorphous carbon films grown on crystalline and nitrogenated silicon substrates by filtered cathodic vacuum arc deposition

Amorphous carbon (a-C) films are widely used as protective overcoats in many technology sectors, principally due to their excellent thermophysical properties and chemical inertness. The growth and thermal stability of sub-5-nm-thick a-C films synthesized by filtered cathodic vacuum arc on pure (crystalline) and nitrogenated (amorphous) silicon substrate surfaces were investigated in this study. Samples of a-C/Si and a-C/SiN x /Si stacks were thermally annealed for various durations and subsequently characterized by high-resolution transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS). The TEM images confirmed the continuity and uniformity of the a-C films and the 5-nm-thick SiN x underlayer formed by silicon nitrogenation using radio-frequency sputtering. The EELS analysis of cross-sectional samples revealed the thermal stability of the a-C films and the efficacy of the SiNx underlayer to prevent carbon migration into the silicon substrate, even after prolonged heating. The obtained results provide insight into the important attributes of an underlayer in heated multilayered media for preventing elemental intermixing with the substrate, while preserving the structural stability of the a-C film at the stack surface. An important contribution of this investigation is the establishment of an experimental framework for accurately assessing the thermal stability and elemental diffusion in layered microstructures exposed to elevated temperatures.

42 ENGINEERING↗

DOE OSTI2022

Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition

Clostridioides difficile is one of the most common causes of antibiotic-associated diarrhea in developed countries. As key virulence factors of C. difficile, toxin A (TcdA) and toxin B (TcdB) act by glucosylating and inactivating Rho and Ras family small GTPases in host cells, which leads to actin cytoskeleton disruption, cell rounding, and ultimately cell death. Here we present the co-crystal structure of the glucosyltransferase domain (GTD) of TcdA in complex with its substrate human RhoA at 2.60-angstrom resolution. This structure reveals that TcdA GTD grips RhoA mainly through its switch I and switch II regions, which is complemented by interactions involving RhoA’s pre-switch I region. Comprehensive structural comparisons between the TcdA GTD–RhoA complex and the structures of TcdB GTD in complex with Cdc42 and R-Ras reveal both the conserved and divergent features of these two toxins in terms of substrate recognition. Taken together, these findings establish the structural basis for TcdA recognition of small GTPases and advance our understanding of the substrates selectivity of large clostridial toxins.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

A novel hardmask-to-substrate pattern transfer method for creating 3D, multi-level, hierarchical, high aspect-ratio structures for applications in microfluidics and cooling technologies

Abstract This letter solves a major hurdle that mars photolithography-based fabrication of micro-mesoscale structures in silicon. Conventional photolithography is usually performed on smooth, flat wafer surfaces to lay a 2D design and subsequently etch it to create single-level features. It is, however, unable to process non-flat surfaces or already etched wafers and create more than one level in the structure. In this study, we have described a novel cleanroom-based process flow that allows for easy creation of such multi-level, hierarchical 3D structures in a substrate. This is achieved by introducing an ultra-thin sacrificial silicon dioxide hardmask layer on the substrate which is first 3D patterned via multiple rounds of lithography. This 3D pattern is then scaled vertically by a factor of 200–300 and transferred to the substrate underneath via a single shot deep etching step. The proposed method is also easily characterizable—using features of different topographies and dimensions, the etch rates and selectivities were quantified; this characterization information was later used while fabricating specific target structures. Furthermore, this study comprehensively compares the novel pattern transfer technique to already existing methods of creating multi-level structures, like grayscale lithography and chip stacking. The proposed process was found to be cheaper, faster, and easier to standardize compared to other methods—this made the overall process more reliable and repeatable. We hope it will encourage more research into hybrid structures that hold the key to dramatic performance improvements in several micro-mesoscale devices.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗