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At least 937 records · Page 52
All sourcesNASA NTRSDOE OSTI
NASA NTRS2003

Effects of carbonyl bond, metal cluster dissociation, and evaporation rates on predictions of nanotube production in high-pressure carbon monoxide

The high-pressure carbon monoxide (HiPco) process for producing single-wall carbon nanotubes (SWNTs) uses iron pentacarbonyl as the source of iron for catalyzing the Boudouard reaction. Attempts using nickel tetracarbonyl led to no production of SWNTs. This paper discusses simulations at a constant condition of 1300 K and 30 atm in which the chemical rate equations are solved for different reaction schemes. A lumped cluster model is developed to limit the number of species in the models, yet it includes fairly large clusters. Reaction rate coefficients in these schemes are based on bond energies of iron and nickel species and on estimates of chemical rates for formation of SWNTs. SWNT growth is measured by the conformation of CO2. It is shown that the production of CO2 is significantly greater for FeCO because of its lower bond energy as compared with that of NiCO. It is also shown that the dissociation and evaporation rates of atoms from small metal clusters have a significant effect on CO2 production. A high rate of evaporation leads to a smaller number of metal clusters available to catalyze the Boudouard reaction. This suggests that if CO reacts with metal clusters and removes atoms from them by forming MeCO, this has the effect of enhancing the evaporation rate and reducing SWNT production. The study also investigates some other reactions in the model that have a less dramatic influence.

Models, Chemical↗
NASA NTRS2009

Multiscale Computer Simulation of Tensile and Compressive Strain in Polymer- Coated Silica Aerogels

While the low thermal conductivities of silica aerogels have made them of interest to the aerospace community as lightweight thermal insulation, the application of conformal polymer coatings to these gels increases their strength significantly, making them potentially useful as structural materials as well. In this work we perform multiscale computer simulations to investigate the tensile and compressive strain behavior of silica and polymer-coated silica aerogels. Aerogels are made up of clusters of interconnected particles of amorphous silica of less than bulk density. We simulate gel nanostructure using a Diffusion Limited Cluster Aggregation (DLCA) procedure, which produces aggregates that exhibit fractal dimensions similar to those observed in real aerogels. We have previously found that model gels obtained via DLCA exhibited stress-strain curves characteristic of the experimentally observed brittle failure. However, the strain energetics near the expected point of failure were not consistent with such failure. This shortcoming may be due to the fact that the DLCA process produces model gels that are lacking in closed-loop substructures, compared with real gels. Our model gels therefore contain an excess of dangling strands, which tend to unravel under tensile strain, producing non-brittle failure. To address this problem, we have incorporated a modification to the DLCA algorithm that specifically produces closed loops in the model gels. We obtain the strain energetics of interparticle connections via atomistic molecular statics, and abstract the collective energy of the atomic bonds into a Morse potential scaled to describe gel particle interactions. Polymer coatings are similarly described. We apply repeated small uniaxial strains to DLCA clusters, and allow relaxation of the center eighty percent of the cluster between strains. The simulations produce energetics and stress-strain curves for looped and nonlooped clusters, for a variety of densities and interaction parameters.

Good, Brian↗
DOE OSTI2021

A CO-to-H{sub 2} Ratio of ≈10{sup -5} toward the Herbig Ae Star HK Ori

Measurements of gas mass in protoplanetary gas disks form the basis for estimating the conditions of planet formation. Among the most important constraints derived from disk diagnostics are the abundances of gas-phase species critical for understanding disk chemistry. Toward this end, we present direct line-of-sight measurements of H{sub 2} and CO, employing UV absorption spectroscopy from Hubble Space Telescope Cosmic Origins Spectrograph to characterize disk composition, molecular excitation temperatures, and spatial distribution in the circumstellar material around the Herbig Ae stars HK Ori and T Ori. We observe strong CO (N(CO) = 10{sup 15.5} cm{sup −2}; T {sub rot}(CO) = 19 K) and H{sub 2} (N(H{sub 2}) = 10{sup 20.34} cm{sup −2}; T {sub rot}(H{sub 2}) = 141 K) absorption toward HK Ori with a CO/H{sub 2} ratio of (≡N(CO)/N(H{sub 2})) = 1.3{sub −0.7}{sup +1.6} × 10{sup −5}. These measurements place direct empirical constraints on the CO-to-H{sub 2} conversion factor in the disk around a Herbig Ae star for the first time, although there is uncertainty concerning the exact viewing geometry of the disk. The spectra of T Ori show CO (N(CO) = 10{sup 14.9} cm{sup −2}; T {sub rot}(CO) = 124 K) absorption. Interestingly, we do not detect any H{sub 2} absorption toward this star (N(H{sub 2}) < 10{sup 15.9} cm{sup −2}). We discuss a potential scenario for the detection of CO without H{sub 2}, which deserves further investigation. The low abundance ratio measured around HK Ori suggests significant depletion of CO in the circumstellar gas, which conforms with the handful of other recent CO abundance measurements in protoplanetary disks.

47 OTHER INSTRUMENTATION↗
DOE OSTI2024

Structural and dynamic changes in P-Rex1 upon activation by PIP 3 and inhibition by IP 4

PIP 3 -dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP 3 and heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP 3 , we discovered that Ins(1,3,4,5)P 4 (IP 4 ) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP 4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH–DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH–DEP1 and PH–4HB interfaces were disturbed exhibited enhanced activity during chemokineinduced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP 3 -containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP 4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2003

Towards the Structure Determination of a Modulated Protein Crystal: The Semicrystalline State of Profilin:Actin

One of the remaining challenges to structural biology is the solution of modulated structures. While small molecule crystallographers have championed this type of structure, to date, no modulated macromolecular structures have been determined. Modulation of the molecular structures within the crystal can produce satellite reflections or a superlattice of reflections in reciprocal space. We have developed the data collection methods and strategies that are needed to collect and analyze these data. If the macromolecule's crystal lattice is composed of physiologically relevant packing contacts, structural changes induced under physiological conditions can cause distortion relevant to the function and biophysical processes of the molecule making up the crystal. By careful measurement of the distortion, and the corresponding three-dimensional structure of the distorted molecule, we will visualize the motion and mechanism of the biological macromolecule(s). We have measured the modulated diffraction pattern produced by the semicrystalline state of profilin:actin crystals using highly parallel and highly monochromatic synchrotron radiation coupled with fine phi slicing (0.001-0.010 degrees) for structure determination. These crystals present these crystals present a unique opportunity to address an important question in structural biology. The modulation is believed to be due to the formation of actin helical filaments from the actin beta ribbon upon the pH-induced dissociation of profilin. To date, the filamentous state of actin has resisted crystallization and no detailed structures are available. The semicrystalline state profilin:actin crystals provides a unique opportunity to understand the many conformational states of actin. This knowledge is essential for understanding the dynamics underlying shape changes and motility of eukaryotic cells. Many essential processes, such as cytokinesis, phagocytosis, and cellular migration depend upon the capacity of the actin microfilament system to be restructured in a controlled manner via polymerization, depolymerization, severing, cross-linking, and anchorage. The structure the semicrystalline state of profilin:actin will challenge and validate current models of muscle contraction and cell motility. The methodology and theory under development will be easily extendable to other systems.

Borgstahl, G.↗
DOE OSTI2023

Genetic encoding of 3–nitro–tyrosine reveals the impacts of 14–3–3 nitration on client binding and dephosphorylation

14–3–3 proteins are central hub regulators of hundreds of phosphorylated “client” proteins. They are subject to over 60 post–translational modifications (PTMs), yet little is known how these PTMs alter 14–3–3 function and its ability to regulate downstream signaling pathways. An often neglected, but well–documented 14–3–3 PTM found under physiological and immune–stimulatory conditions is the conversion of tyrosine to 3–nitro–tyrosine at several Tyr sites, two of which are located at sites considered important for 14–3–3 function: Y130 (β–isoform numbering) is located in the primary phospho–client peptide–binding groove, while Y213 is found on a secondary binding site that engages with clients for full 14–3–3/client complex formation and client regulation. By genetically encoding 3–nitro–tyrosine, we sought to understand if nitration at Y130 and Y213 effectively modulated 14–3–3 structure, function, and client complexation. The 1.5 Å resolution crystal structure of 14–3–3 nitrated at Y130 showed the nitro group altered the conformation of key residues in the primary binding site, while functional studies confirmed client proteins failed to bind this variant of 14–3–3. But, in contrast to other client–binding deficient variants, it did not localize to the nucleus. Further, the 1.9 Å resolution structure of 14–3–3 nitrated at Y213 revealed unusual flexibility of its C–terminal α–helix resulting in domain swapping, suggesting additional structural plasticity though its relevance is not clear as this nitrated form retained its ability to bind clients. Collectively, our data suggest that nitration of 14–3–3 will alter downstream signaling systems, and if uncontrolled could result in global dysregulation of the 14–3–3 interactome.

14-3-3↗
DOE OSTI2023

Entangled spin-polarized excitons from singlet fission in a rigid dimer

Abstract Singlet fission, a process that splits a singlet exciton into a biexciton, has promise in quantum information. We report time-resolved electron paramagnetic resonance measurements on a conformationally well-defined acene dimer molecule, TIPS-BP1', designed to exhibit strongly state-selective relaxation to specific magnetic spin sublevels. The resulting optically pumped spin polarization is a nearly pure initial state from the ensemble. The long-lived spin coherences modulate the signal intrinsically, allowing a measurement scheme that substantially removes noise and uncertainty in the magnetic resonance spectra. A nonadiabatic transition theory with a minimal number of spectroscopic parameters allows the quantitative assignment and interpretation of the spectra. In this work, we show that the rigid dimer TIPS-BP1' supports persistent spin coherences at temperatures far higher than those used in conventional superconducting quantum hardware.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗
DOE OSTI2022

The role of filamentation in activation and DNA sequence specificity of the sequence-specific endonuclease SgrAI

Filament formation by metabolic, biosynthetic, and other enzymes has recently come into focus as a mechanism to fine-tune enzyme activity in the cell. Filamentation is key to the function of SgrAI, a sequence-specific DNA endonuclease that has served as a model system to provide some of the deepest insights into the biophysical characteristics of filamentation and its functional consequences. Structure-function analyses reveal that, in the filamentous state, SgrAI stabilizes an activated enzyme conformation that leads to accelerated DNA cleavage activity and expanded DNA sequence specificity. The latter is thought to be mediated by sequence-specific DNA structure, protein–DNA interactions, and a disorder-to-order transition in the protein, which collectively affect the relative stabilities of the inactive, non-filamentous conformation and the active, filamentous conformation of SgrAI bound to DNA. Full global kinetic modeling of the DNA cleavage pathway reveals a slow, rate-limiting, second-order association rate constant for filament assembly, and simulations of in vivo activity predict that filamentation is superior to non-filamenting mechanisms in ensuring rapid activation and sequestration of SgrAI's DNA cleavage activity on phage DNA and away from the host chromosome. In vivo studies demonstrate the critical requirement for accelerated DNA cleavage by SgrAI in its biological role to safeguard the bacterial host. Collectively, these data have advanced our understanding of how filamentation can regulate enzyme structure and function, while the experimental strategies used for SgrAI can be applied to other enzymatic systems to identify novel functional roles for filamentation.

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

Human cytochrome P450 17A1 structures with metabolites of prostate cancer drug abiraterone reveal substrate-binding plasticity and a second binding site

Abiraterone acetate is a first-line therapy for castration-resistant prostate cancer. This prodrug is deacetylated in vivo to abiraterone, which is a potent and specific inhibitor of cytochrome P450 17A1 (CYP17A1). CYP17A1 performs two sequential steps that are required for the biosynthesis of androgens that drive prostate cancer proliferation, analogous to estrogens in breast cancer. Abiraterone can be further metabolized in vivo on the steroid A ring to multiple metabolites that also inhibit CYP17A1. Despite its design as an active-site-directed substrate analog, abiraterone and its metabolites demonstrate mixed competitive/noncompetitive inhibition. To understand their binding, we solved the X-ray structures of CYP17A1 with three primary abiraterone metabolites. Despite different conformations of the steroid A ring and substituents, all three bound in the CYP17A1 active site with the steroid core packed against the I helix and the A ring C3 keto or hydroxyl oxygen forming a hydrogen bond with N202 similar to abiraterone itself. The structure of CYP17A1 with 3-keto, 5α-abiraterone was solved to 2.0 Å, the highest resolution to date for a CYP17A1 complex. This structure had additional electron density near the F/G loop, which is likely a second molecule of the inhibitor and which may explain the noncompetitive inhibition. Mutation of the adjacent Asn52 to Tyr positions its side chain in this space, maintains enzyme activity, and prevents binding of the peripheral ligand. Collectively, our findings provide further insight into abiraterone metabolite binding and CYP17A1 function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative Pore Structure and Dynamics for Bacterial Microcompartment Shell Protein Assemblies in Sheets or Shells

Bacterial microcompartments (BMCs) are protein-bound organelles found in some bacteria that encapsulate enzymes for enhanced catalytic activity. These compartments spatially sequester enzymes within semipermeable shell proteins, analogous to many membrane-bound organelles. The shell proteins assemble into multimeric tiles; hexamers, trimers, and pentamers, and these tiles self-assemble into larger assemblies with icosahedral symmetry. While icosahedral shells are the predominant form in vivo , the tiles can also form nanoscale cylinders or sheets. The individual multimeric tiles feature central pores that are key to regulating transport across the protein shell. Our primary interest is to quantify pore shape changes in response to alternative component morphologies at the nanoscale. We used molecular modeling tools to develop atomically detailed models for both planar sheets of tiles and curved structures representative of the complete shells found in vivo . Subsequently, these models were animated using classical molecular dynamics simulations. From the resulting trajectories, we analyzed the overall structural stability, water accessibility to individual residues, water residence time, and pore geometry for the hexameric and trimeric protein tiles from the Haliangium ochraceu m model BMC shell. These exhaustive analyses suggest no substantial variation in pore structure or solvent accessibility between the flat and curved shell geometries. We additionally compare our analysis to hydroxyl radical footprinting data to serve as a check against our simulation results, highlighting specific residues where water molecules are bound for a long time. Although with little variation in morphology or water interaction, we propose that the planar and capsular morphology can be used interchangeably when studying permeability through BMC pores.

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

Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus

Abstract Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Predicting and Understanding Non-Covalent Interactions Using Novel Forms of Symmetry-Adapted Perturbation Theory

Although sometimes derided as “weak” interactions, non-covalent forces play a critical role in ligand binding and crystal packing and in determining the conformational landscape of flexible molecules. Symmetry-adapted perturbation theory (SAPT) provides a framework for accurate ab initio calculation of intermolecular interactions and furnishes a natural decomposition of the interaction energy into physically meaningful components: semiclassical electrostatics (rigorously obtained from monomer charge densities), Pauli or steric repulsion, induction (including both polarization and charge transfer), and dispersion. This decomposition helps to foster deeper understanding of non-covalent interactions and can be used to construct transferable, physics-based force fields. Separability of the SAPT interaction energy also provides the flexibility to construct composite methods, a feature that we exploit to improve the description of dispersion interactions. Furthermore, these are challenging to describe accurately because they arise from nonlocal electron correlation effects that appear for the first time at second order in perturbation theory but are not quantitatively described at that level.

74 ATOMIC AND MOLECULAR PHYSICS↗
DOE OSTI2024

Design of amyloidogenic peptide traps

Segments of proteins with high β-strand propensity can self-associate to form amyloid fibrils implicated in many diseases. We describe a general approach to bind such segments in β-strand and β-hairpin conformations using de novo designed scaffolds that contain deep peptide-binding clefts. The designs bind their cognate peptides in vitro with nanomolar affinities. The crystal structure of a designed protein–peptide complex is close to the design model, and NMR characterization reveals how the peptide-binding cleft is protected in the apo state. We use the approach to design binders to the amyloid-forming proteins transthyretin, tau, serum amyloid A1 and amyloid β 1–42 (Aβ42). The Aβ binders block the assembly of Aβ fibrils as effectively as the most potent of the clinically tested antibodies to date and protect cells from toxic Aβ42 species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Electron Sextets as Optically Addressable Molecular Qubits: Triplet Carbenes

There is a growing demand in quantum information science and sensing for electron spin purification and readout via a spin-optical interface. This technique, known as optically detected magnetic resonance (ODMR), has been applied to diamond-NV centers and transition-metal complexes. Metal-free counterparts of these optically addressable spin qubits promise to be cheaper, more sustainable color centers with prolonged polarization lifetimes. However, progress has been hindered by the low ODMR signals of carbon-based π-diradicals, partly due to the lack of a ground singlet-to-triplet intersystem crossing (ISC). In this work, we propose exploring organic systems that are even more electron-deficient: electron sextets. Using triplet carbenes as an example, we illustrate how the ground singlet-triplet gap can be widened beyond thermal energy with the associated singlet-to-triplet ISC made available by vibronic effects. Through careful molecular engineering, this ISC can occur at a rate similar to and with an opposite spin selectivity from the excited-state ISC well-established in π-diradicals, unlocking a new ODMR pathway with potential signal gains. Persistent triplet carbenes are a renascent field, with multiple stable molecules being isolated in the past five years. To motivate further development of its emissive properties, we illustrate our design in three realistic carbene candidates that incorporate existing strategies for carbene stabilization. Furthermore, we believe that a new realm of quantum materials can be uncovered by expanding our scope toward stable electron sextets.

Carbene compounds↗
DOE OSTI2023

Use of Stable Mercury Isotopes to Assess Mercury and Methylmercury Transformation and Transport across Critical Interfaces from the Molecular to the Watershed Scale (Final Report)

This project titled “Use of Stable Mercury Isotopes to Assess Mercury and Methylmercury Transformation and Transport across Critical Interfaces from the Molecular to the Watershed Scale” represents a collaborative effort between the University of Michigan (Jason Demers, PI) and Oak Ridge National Laboratory (Scott Brooks, co-I). Much has been learned about mercury (Hg) cycling in stream ecosystems, and East Fork Poplar Creek (EFPC) in particular, through decades of previous research. Nevertheless, some of the most fundamental questions regarding the sources of bioavailable Hg and its transformation to toxic methylmercury (MeHg) have remained unanswered. These fundamental questions include: (1) what are the sources and biogeochemical processes that lead to the input of dissolved Hg to stream water across critical subsurface interfaces within stream ecosystems, and EFPC in particular? and (2) what are the sources and biogeochemical processes that control the production and fate of bioaccumulative MeHg within stream ecosystems, and in EFPC in particular? To address these fundamental questions, our project aimed to couple laboratory experiments and field observations, both utilizing natural abundance Hg stable isotope techniques, to identify the processes responsible for generating mobile, bioavailable dissolved Hg from recalcitrant legacy sources within critical subsurface zones (e.g., streambed hyporheic zone, riparian floodplain subsurface). We used the isotopic signature of this bioavailable dissolved Hg to track its mobilization across these critical interfaces in order to link diffuse subsurface sources of dissolved Hg with increases in surface water dissolved Hg flux measured at the watershed scale. Additionally, our research aimed to determine the isotopic composition of MeHg within these same critical subsurface zones. We directly assessed the isotopic composition of MeHg within biota in order to gain insight into which subsurface sources of inorganic Hg and toxic MeHg are available for bioaccumulation within the EFPC ecosystem. Net fluxes of dissolved Hg along the flow path of EFPC were shown to vary spatially and temporally. In the Upper EFPC, within the Y12 boundary, stream water flux of dissolved Hg consistently decreased between the outfall and the downstream boundary of Y12 (57% ± 29%, 1SD). Within the Upper EFPC, an assessment of Hg isotopic composition suggested that losses were strongly reaction-driven, although isotopic diagnostics did not conform to any known processes. Downstream of Y12, in the upper reach of the Lower EFPC, dissolved Hg fluxes tended to increase during the dormant season (net gain of 11-120%), and decrease during the growing season (net loss of 23% +/- 18%, 1SD). In the downstream-most reach of Lower EFPC, dissolved Hg fluxes increased by 12-108% in 9 out of 10 monthly assessments. Overall, diffuse fluxes from the non-Y12 watershed accounted for 34% (+/- 17%, 1SD) of all dissolved Hg exported during base flow. Within Lower EFPC, an assessment of Hg isotopic composition was consistent with the contribution of diffuse Hg inputs from high-concentration hotspots within riparian floodplains and streambed hyporheic pore water. To investigate remobilization of recalcitrant Hg from legacy sediment sources, we developed procedures that coupled isotopic analysis with sequential extractions of streambed sediment. We found that the proportion of weakly-bound Hg within EFPC streambed sediment was relatively small, but could still account for a large proportion of the annual flux of dissolved Hg from EFPC. These sequential extractions also showed that this weakly-bound Hg fraction could be replenished from the much larger fraction of recalcitrant Hg in sediment. The isotopic composition of these weakly-bound and remobilized recalcitrant Hg fractions within the sediment was consistent with high-concentration dissolved Hg hotspots within hyporheic pore water. Thus, this research provided novel evidence that legacy mercury sources within streambed sediment could provide an ongoing contribution of dissolved Hg to surface waters. Finally, we developed new methods for the direct determination of the MeHg isotopic composition of organisms, which allowed a more direct evaluation of inorganic Hg and MeHg sources accumulating in the food web. We found that fish and aquatic invertebrates in both EFPC and a regional background site obtained inorganic Hg and MeHg from multiple isotopically distinct sources, including sediment, suspended particulates, and periphyton. Photodemethylation was found to be an important reaction influencing MeHg dynamics at both sites. However, the balance of microbial methylation and demethylation processes differed between the two streams, with fractionation resulting from methylation and demethylation processes being relatively in balance within the regional background site, whereas microbial methylation appeared to be dominant over microbial demethylation within the EFPC ecosystem. Broadly, the application of Hg isotopic analysis in this study led to numerous novel insights regarding the biogeochemical cycling of Hg in stream ecosystems. This research project promoted the development of two new approaches, including the coupling of sequential extractions with Hg isotopic analysis to assess remobilization of recalcitrant Hg within sediments, and a new method for the isotopic analysis of MeHg isolated from environmental samples. Both of these efforts represent advances in capacity for the field of mercury isotopic analysis and environmental assessment. Overall, this study demonstrates that the application of Hg stable isotope techniques continues to provide new insights into the biogeochemical cycling of Hg in complex aquatic environments, both within the EFPC and beyond.

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

Going around the Kok cycle of the water oxidation reaction with femtosecond X-ray crystallography

The water oxidation reaction in photosystem II (PS II) produces most of the molecular oxygen in the atmosphere, which sustains life on Earth, and in this process releases four electrons and four protons that drive the downstream process of CO 2 fixation in the photosynthetic apparatus. The catalytic center of PS II is an oxygen-bridged Mn 4 Ca complex (Mn 4 CaO 5 ) which is progressively oxidized upon the absorption of light by the chlorophyll of the PS II reaction center, and the accumulation of four oxidative equivalents in the catalytic center results in the oxidation of two waters to dioxygen in the last step. The recent emergence of X-ray free-electron lasers (XFELs) with intense femtosecond X-ray pulses has opened up opportunities to visualize this reaction in PS II as it proceeds through the catalytic cycle. In this review, we summarize our recent studies of the catalytic reaction in PS II by following the structural changes along the reaction pathway via room-temperature X-ray crystallography using XFELs. The evolution of the electron density changes at the Mn complex reveals notable structural changes, including the insertion of O X from a new water molecule, which disappears on completion of the reaction, implicating it in the O—O bond formation reaction. We were also able to follow the structural dynamics of the protein coordinating with the catalytic complex and of channels within the protein that are important for substrate and product transport, revealing well orchestrated conformational changes in response to the electronic changes at the Mn 4 Ca cluster.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2024

Structural and biochemical characterization of the mitomycin C repair exonuclease MrfB

Mitomycin C (MMC) repair factor A (mrfA) and factor B (mrfB), encode a conserved helicase and exonuclease that repair DNA damage in the soil-dwelling bacterium Bacillus subtilis. Here we have focused on the characterization of MrfB, a DEDDh exonuclease in the DnaQ superfamily. We solved the structure of the exonuclease core of MrfB to a resolution of 2.1 Å, in what appears to be an inactive state. In this conformation, a predicted α-helix containing the catalytic DEDDh residue Asp172 adopts a random coil, which moves Asp172 away from the active site and results in the occupancy of only one of the two catalytic Mg 2+ ions. We propose that MrfB resides in this inactive state until it interacts with DNA to become activated. By comparing our structure to an AlphaFold prediction as well as other DnaQ-family structures, we located residues hypothesized to be important for exonuclease function. Using exonuclease assays we show that MrfB is a Mg 2+ -dependent 3'–5' DNA exonuclease. We show that Leu113 aids in coordinating the 3' end of the DNA substrate, and that a basic loop is important for substrate binding. This work provides insight into the function of a recently discovered bacterial exonuclease important for the repair of MMC-induced DNA adducts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Angle between DNA linker and nucleosome core particle regulates array compaction revealed by individual-particle cryo-electron tomography

Abstract The conformational dynamics of nucleosome arrays generate a diverse spectrum of microscopic states, posing challenges to their structural determination. Leveraging cryogenic electron tomography (cryo-ET), we determine the three-dimensional (3D) structures of individual mononucleosomes and arrays comprising di-, tri-, and tetranucleosomes. By slowing the rate of condensation through a reduction in ionic strength, we probe the intra-array structural transitions that precede inter-array interactions and liquid droplet formation. Under these conditions, the arrays exhibite irregular zig-zag conformations with loose packing. Increasing the ionic strength promoted intra-array compaction, yet we do not observe the previously reported regular 30-nanometer fibers. Interestingly, the presence of H1 do not induce array compaction; instead, one-third of the arrays display nucleosomes invaded by foreign DNA, suggesting an alternative role for H1 in chromatin network construction. We also find that the crucial parameter determining the structure adopted by chromatin arrays is the angle between the entry and exit of the DNA and the corresponding tangents to the nucleosomal disc. Our results provide insights into the initial stages of intra-array compaction, a critical precursor to condensation in the regulation of chromatin organization.

59 BASIC BIOLOGICAL SCIENCES↗