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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 163 records · Page 9
All sourcesNASA NTRSDOE OSTI
DOE OSTI2018

Advances in the Recovery of Uranium from Seawater: Studies Under Real Ocean Conditions

The objective of this project was to move the testing of uranium adsorbents from the laboratory to the field. We were successfully able to test fibers in both a flume and ocean environment under varying conditions to quantify the adsorptive properties and uranium uptake of adsorbents under coastal ocean conditions. Through this testing we have identified numerous challenges to be overcome prior to large-scale deployment of these fibers This project began in Phase I with laboratory experiments examining the effects of flow rates and water filtration on the absorption of uranium to fibers in flowthrough columns. In this experiment we found that fibers adsorb most efficiently at a flow rate of 250 ml/min, on a per liter basis. However faster flow rates increase exposure volume and allowed for slightly greater total U absorption. Therefore a balance between current speeds that are too fast and potentially break down fibers and those that are slower will be important to consider, alongside bio-fouling and exposure time, when deciding which current regimes are best to place fibers in the ocean. Phase II examined biofouling, comparing exposure of fibers in filtered flume water to that in a coastal ocean environment. Results from the dock and flume parallel experiment indicate a strong effect of biofouling on the capacity of the adsorbent fibers to adsorb uranium towards the very end of the experiment. Dock and flume samples showed comparable adsorption rates with the flume maximum achieved at day 49 with an adsorbance of 3.4 g U/kg-ads while the dock maximum was reached at day 42 with an adsorbance of 2.7 g U/kg-ads. Biofouling mitigation techniques were also examined. A comparison of 5 m and 12 m samples showed less growth at 12 meters where there is less light as well as less growth on the copper cages, which are toxic to many marine organisms. Overall despite biofouling, particles and other elements present, the fibers adsorbed uranium up to 2.7 g U/kg-ads in our open water dock experiments. This is lower than the maximum achieved in the flume of 3.4 g U/kg-ads and much lower than rates observed with synthetic seawater in laboratory experiments. Phase III involved two parts: working at Woods Hole Oceanographic Institution (WHOI) we examined the feasibility of reusing fibers after ocean deployment. Fiber reuse is an important consideration for operational costs associated with deployment. At present our data implies that reuse after open ocean exposure may not be significantly more effective than a single long deployment of fibers. The second part of Phase III involved collaboration with A. Slocum and M. Haji at MIT to examine the effects of various enclosures for fibers. This also involved testing a rotating system, designed to be deployed as part of an offshore wind turbine.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
DOE OSTI2022

Modeling receptor flexibility in the structure-based design of KRAS G12C inhibitors

KRAS has long been referred to as an ‘undruggable’ target due to its high affinity for its cognate ligands (GDP and GTP) and its lack of readily exploited allosteric binding pockets. Recent progress in the development of covalent inhibitors of KRAS G12C has revealed that occupancy of an allosteric binding site located between the α3-helix and switch-II loop of KRAS G12C —sometimes referred to as the ‘switch-II pocket’—holds great potential in the design of direct inhibitors of KRAS G12C . In studying diverse switch-II pocket binders during the development of sotorasib (AMG 510), the first FDA-approved inhibitor of KRAS G12C , we found the dramatic conformational flexibility of the switch-II pocket posing significant challenges toward the structure-based design of inhibitors. Here, we present our computational approaches for dealing with receptor flexibility in the prediction of ligand binding pose and binding affinity. For binding pose prediction, we modified the covalent docking program CovDock to allow for protein conformational mobility. This new docking approach, termed as FlexCovDock, improves success rates from 55 to 89% for binding pose prediction on a dataset of 10 cross-docking cases and has been prospectively validated across diverse ligand chemotypes. For binding affinity prediction, we found standard free energy perturbation (FEP) methods could not adequately handle the significant conformational change of the switch-II loop. We developed a new computational strategy to accelerate conformational transitions through the use of targeted protein mutations. Using this methodology, the mean unsigned error (MUE) of binding affinity prediction were reduced from 1.44 to 0.89 kcal/mol on a set of 14 compounds. These approaches were of significant use in facilitating the structure-based design of KRAS G12C inhibitors and are anticipated to be of further use in the design of covalent (and noncovalent) inhibitors of other conformationally labile protein targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Assessing the impact of substrate-level enzyme regulations limiting ethanol titer in Clostridium thermocellum using a core kinetic model

Clostridium thermocellum is a promising candidate for consolidated bioprocessing because it can directly ferment cellulose to ethanol. Despite significant efforts, achieved yields and titers fall below industrially relevant targets. This implies that there still exist unknown enzymatic, regulatory, and/or possibly thermodynamic bottlenecks that can throttle back metabolic flow. By (i) elucidating internal metabolic fluxes in wild-type C. thermocellum grown on cellobiose via 13 C-metabolic flux analysis ( 13 C-MFA), (ii) parameterizing a core kinetic model, and (iii) subsequently deploying an ensemble-docking workflow for discovering substrate-level regulations, this paper aims to reveal some of these factors and expand our knowledgebase governing C. thermocellum metabolism. Generated 13 C labeling data were used with 13 C-MFA to generate a wild-type flux distribution for the metabolic network. Notably, flux elucidation through MFA alluded to serine generation via the mercaptopyruvate pathway. Using the elucidated flux distributions in conjunction with batch fermentation process yield data for various mutant strains, we constructed a kinetic model of C. thermocellum core metabolism (i.e. k-ctherm138). Subsequently, we used the parameterized kinetic model to explore the effect of removing substrate-level regulations on ethanol yield and titer. Upon exploring all possible simultaneous (up to four) regulation removals we identified combinations that lead to many-fold model predicted improvement in ethanol titer. In addition, by coupling a systematic method for identifying putative competitive inhibitory mechanisms using K-FIT kinetic parameterization with the ensemble-docking workflow, we flagged 67 putative substrate-level inhibition mechanisms across central carbon metabolism supported by both kinetic formalism and docking analysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Modeling Protein–Protein and Protein–Ligand Interactions by the ClusPro Team in CASP16

ABSTRACT In the CASP16 experiment, our team employed hybrid computational strategies to predict both protein–protein and protein–ligand complex structures. For protein–protein docking, we combined physics‐based sampling—using ClusPro FFT docking and molecular dynamics—with AlphaFold (AF)‐based sampling, followed by AF‐based refinement. Our method produced numerous high‐accuracy complex models, including cases where AF alone failed, underscoring the critical role of physics‐based sampling alongside deep learning‐based refinement. For protein–ligand docking, we integrated the ClusPro LigTBM template‐based approach with a machine learning‐based confidence model for rescoring. The method preserves conserved interaction fragments derived from homologous complexes, followed by local resampling using physics‐based sampling and a diffusion model. Our template‐based strategy achieved a mean lDDT‐PLI of 0.69 across 233 targets, which was highly competitive. These results demonstrate that combining physics‐based modeling with AI‐driven refinement can significantly enhance the accuracy of both protein–protein and protein–ligand structure predictions.

Ashizawa, Ryota [Department of Applied Mathematics↗
DOE OSTI2019

Structure-based lead optimization to improve the antifungal potency of the tetrahydroimidazo pyridine inhibitors targeted to Candida albicans dihydrofolate reductase and lanosterol 14-alpha-demethylase

We investigate the binding and inhibition profiles for a selected dataset of tetrahydroimidazo pyridine molecules against Candida albicans dihydrofolate reductase (DHFR) and lanosterol 14-alpha-demethylase (CYP51). A hit molecule was screened and identified through Lipinski’s rule of five, ADMET (absorption, distribution, metabolism, excretion, and toxicity), and the molecular docking. Some inhibitors of our design have shown positive drug scores, good solubilities, and high docking scores over the selected dataset. The first-principles calculation based on the density functional theory was carried out to understand how the electronic distributions of frontier orbitals of molecules affect their inhibition profiles. The present structure-based approach enabled us to design new pharmacophore analogs with improved ADMET profile, drug scores, and docking scores against selected receptors. .

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Metabolism of bilirubin by human cytochrome P450 2A6

The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic CYP2A6 protein and activity levels but not mRNA. ► Co-treatment with a protein synthesis inhibitor prolongs CYP2A6 half-life.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

Rationale: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca{sup 2+} channels and their renowned antioxidant properties. Methods: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca{sup 2+} channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca{sup 2+} channel-blocking activity and antioxidant properties. The Ca{sup 2+} channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Results: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca{sup 2+} channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca{sup 2+} channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Conclusions: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. - Highlights: • Dihydropyridine (DHP) molecules are widely used in cardiovascular disease. • DHPs block Ca{sup 2+} entry through LTCC—some DHPs have antioxidant activity as well. • We synthesized 6 new DHPs and tested their Ca{sup 2+} blocking and antioxidant activities. • 3-Aryl meta-hydroxyl substitution strongly increases their Ca{sup 2+} blocking activity. • 3-Aryl meta-hydroxyl substitution did not affect the antioxidant properties.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R{sup 2} = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict binding affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R{sup 2} = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables prediction of both affinity and efficacy. • Models can be used to identify environmental chemicals interacting with THRβ. • Models can be used to eliminate the THRβ-mediated mechanism of action.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2010

Conserved Binding Mode of Human [beta subscript 2] Adrenergic Receptor Inverse Agonists and Antagonist Revealed by X-ray Crystallography

G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the {beta}{sub 2} adrenergic receptor ({beta}{sub 2}AR) is one of the most extensively studied. Previously, the X-ray crystal structure of {beta}{sub 2}AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered {beta}{sub 2}AR construct in complex with two inverse agonists: ICI 118,551 (2.8 {angstrom}), a recently described compound (2.8 {angstrom}) (Kolb et al, 2009), and the antagonist alprenolol (3.1 {angstrom}). The structures show the same overall fold observed for the previous {beta}{sub 2}AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with {beta}{sub 2}AR. Furthermore, receptor ligand cross-docking experiments revealed that a single {beta}{sub 2}AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.

08 HYDROGEN↗
DOE OSTI2016

Enhanced hydrogenation activity and diastereomeric interactions of methyl pyruvate co-adsorbed with R-1-(1-naphthyl)ethylamine on Pd(111)

Unmodified racemic sites on heterogeneous chiral catalysts reduce their overall enantioselectivity, but this effect is mitigated in the Orito reaction (methyl pyruvate (MP) hydrogenation to methyl lactate) by an increased hydrogenation reactivity. Here, this effect is explored on a R-1-(1-naphthyl)ethylamine (NEA)-modified Pd(111) model catalyst where temperature-programmed desorption experiments reveal that NEA accelerates the rates of both MP hydrogenation and H/D exchange. NEAþMP docking complexes are imaged using scanning tunneling microscopy supplemented by density functional theory calculations to allow the most stable docking complexes to be identified. The results show that diastereomeric interactions between NEA and MP occur predominantly by binding of the C=C of the enol tautomer of MP to the surface, while simultaneously optimizing C=O...H 2 N hydrogen-bonding interactions. In conclusion, the combination of chiral-NEA driven diastereomeric docking with a tautomeric preference enhances the hydrogenation activity since C=C bonds hydrogenate more easily than C=O bonds thus providing a rationale for the catalytic observations.

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

An artificial intelligence accelerated virtual screening platform for drug discovery

Abstract Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel Na V 1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to Na V 1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Science & Technology - Other Topics↗
DOE OSTI2019

Functional divergence caused by mutations in an energetic hotspot in ERK2

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila , only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Overall, our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Combinatorial diversity of Syk recruitment driven by its multivalent engagement with FcεRIγ

Syk/Zap70 family kinases are essential for signaling via multichain immune-recognition receptors such as tetrameric (αβγ2) FcεRI. Syk activation is generally attributed to cis binding of its tandem SH2 domains to dual phosphotyrosines within FcεRIγ-ITAMs (immunoreceptor tyrosine-based activation motifs). However, the mechanistic details of Syk docking on γ homodimers are unresolved. Here, we estimate that multivalent interactions for WT Syk improve cis-oriented binding by three orders of magnitude. We applied molecular dynamics (MD), hybrid MD/worm-like chain polymer modeling, and live cell imaging to evaluate relative binding and signaling output for all possible cis and trans Syk–FcεRIγ configurations. Syk binding is likely modulated during signaling by autophosphorylation on Y130 in interdomain A, since a Y130E phosphomimetic form of Syk is predicted to lead to reduced helicity of interdomain A and alter Syk’s bias for cis binding. Experiments in reconstituted γ-KO cells, whose γ subunits are linked by disulfide bonds, as well as in cells expressing monomeric ITAM or hemITAM γ-chimeras, support model predictions that short distances between γ ITAM pairs are required for trans docking. We propose that the full range of docking configurations improves signaling efficiency by expanding the combinatorial possibilities for Syk recruitment, particularly under conditions of incomplete ITAM phosphorylation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Structure of the lutein-binding domain of human StARD3 at 1.74 Å resolution and model of a complex with lutein

A crystal structure of the lutein-binding domain of human StARD3 (StAR-related lipid-transfer protein 3; also known as MLN64) has been refined to 1.74 Å resolution. A previous structure of the same protein determined to 2.2 Å resolution highlighted homology with StARD1 and shared cholesterol-binding character. StARD3 has since been recognized as a carotenoid-binding protein in the primate retina, where its biochemical function of binding lutein with specificity appears to be well suited to recruit this photoprotective molecule. The current and previous structures correspond closely to each other (r.m.s.d. of 0.25 Å), especially in terms of the helix-grip fold constructed around a solvent-filled cavity. Regions of interest were defined with alternate conformations in the current higher-resolution structure, including Arg351 found within the cavity and Ω1, a loop of four residues found just outside the cavity entrance. Models of the complex with lutein generated by rigid-body docking indicate that one of the ionone rings must protrude outside the cavity, and this insight has implications for molecular interactions with transport proteins and enzymes that act on lutein. Interestingly, models with the ϵ-ionone ring characteristic of lutein pointing towards the bottom of the cavity were associated with fewer steric clashes, suggesting that steric complementarity and ligand asymmetry may play a role in discriminating lutein from the other ocular carotenoids zeaxanthin and meso -zeaxanthin, which only have β-ionone rings.

Biochemistry & Molecular Biology↗
DOE OSTI2015

WE-EF-BRA-01: A Dual-Use Optical Tomography System for Small Animal Radiation Research Platform (SARRP)

Purpose: We develop a novel dual-use configuration for a tri-modality, CBCT/bioluminescence tomography(BLT)/fluorescence tomography(FT), imaging system with the SARRP that can function as a standalone system for longitudinal imaging research and on-board the SARRP to guide irradiation. BLT provides radiation guidance for soft tissue target, while FT offers functional information allowing mechanistic investigations. Methods: The optical assembly includes CCD camera, lens, filter wheel, 3-way mirrors, scanning fiber system and light-tight enclosure. The rotating mirror system directs the optical signal from the animal surface to the camera at multiple projection over 180 degree. The fiber-laser system serves as the external light source for the FT application. Multiple filters are used for multispectral imaging to enhance localization accuracy using BLT. SARRP CBCT provides anatomical information and geometric mesh for BLT/FT reconstruction. To facilitate dual use, the 3-way mirror system is cantilevered in front of the camera. The entire optical assembly is driven by a 1D linear stage to dock onto an independent mouse support bed for standalone application. After completion of on-board optical imaging, the system is retracted from the SARRP to allow irradiation of the mouse. Results: A tissue-simulating phantom and a mouse model with a luminescence light source are used to demonstrate the function of the dual-use optical system. Feasibility data have been obtained based on a manual-docking prototype. The center of mass of light source determined in living mouse with on-board BLT is within 1±0.2mm of that with CBCT. The performance of the motorized system is expected to be the same and will be presented. Conclusion: We anticipate the motorized dual use system provide significant efficiency gain over our manual docking and off-line system. By also supporting off-line longitudinal studies independent of the SARRP, the dual-use system is a highly efficient and cost-effective platform to facilitate optical imaging for pre-clinical radiation research. The work is supported by NIH R01CA158100 and Xstrahl Ltd. Drs. John Wong and Iulian Iordachita receive royalty payment from a licensing agreement between Xstrahl Ltd and Johns Hopkins University. John Wong also has a consultant agreement with Xstrahl Ltd.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

The Cas9 endonuclease is widely used for genome engineering applications by programming its single-guide RNA, and ongoing work is aimed at improving the accuracy and efficiency of DNA targeting. DNA cleavage of Cas9 is controlled by the conformational state of the HNH nuclease domain, but the mechanism that governs HNH activation at on-target DNA while reducing cleavage activity at off-target sites remains poorly understood. Using single-molecule Förster resonance energy transfer, we identified an intermediate state of Streptococcus pyogenes Cas9, representing a conformational checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking into its active state. HNH docking requires divalent cations, but not strand scission, and this docked conformation persists following DNA cleavage. Sequence mismatches between the DNA target and guide RNA prevent transitions from the checkpoint intermediate to the active conformation, providing selective avoidance of DNA cleavage at stably bound off-target sites.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Dose Modeling for Harborview Research and Training Facility

The Harborview Research and Training (R&T) Facility at the University of Washington (UW) was accidently contaminated from a breached sealed source of Cesium-137. The release occurred during removal of the sealed source from its holder within a portable hot cell in the loading dock area near room 220. The loading dock and northeast corner of the building above the ground floor were impacted by the release, whereas the remaining portions of the R&T Facility were minimally affected. Detailed descriptions of the R&T Facility and the affected area/rooms are included in Section 2. The RESRAD Team in the Environmental Science Division of Argonne National Laboratory visited the site on August 12, 2019, and interviewed the R&T Facility building manager and staff working in the building at that time. Site- and building-specific data were collected including building floor plan CAD drawings, building occupants’ working hours, contaminated rooms photos and their dimensions, building air exchange rates, building materials and “curtain wall” details, and so forth. These data are being used in the dose modeling for potential occupants of the building after it is remediated. The dose modeling was performed using the RESRAD-BUILD computer code, which is designed for the evaluation of radiologically contaminated buildings (Yu et al. 2003). Two hypothetical building occupants of the Harborview R&T Facility were evaluated: a researcher occupying room 726 (a research laboratory on the seventh floor) and a custodian occupying room 220 (loading dock and trash compactor room with a rollup door). A maintenance worker that will be spending less time in impacted areas was not modeled because it will be bounded by other two hypothetical building occupants modeled. A summary description of the RESRAD-BUILD is provided in Appendix A, and the modeling of potential doses to a researcher and a custodian is described in detail in Section 3. A summary and a discussion of the dose modeling results are included in Section 4. The parameters used in the RESRAD-BUILD modeling are included in Appendix B. This report documents the dose modeling results; it does not describe measurements of the actual radioactivity levels at the Harborview R&T Facility, nor does it set any expectation for the actual final radioactivity levels. The radiation doses presented are modeling results based on the generic screening values of Cs-137 and several conservative assumptions and are meant only to assist in decision making regarding decontamination of the Harborview R&T Facility.

61 RADIATION PROTECTION AND DOSIMETRY↗
DOE OSTI2012

Dissecting the cAMP-inducible allosteric switch in protein kinase A RI alpha

The regulatory subunits of cAMP-dependent protein kinase (PKA) are the major receptors of cAMP in most eukaryotic cells. As the cyclic nucleotide binding (CNB) domains release cAMP and bind to the catalytic subunit of PKA, they undergo a major conformational change. The change is mediated by the B/C helix in CNB-A, which extends into one long helix that now separates the two CNB domains and docks onto the surface of the catalytic subunit. We explore here the role of three key residues on the B/C helix that dock onto the catalytic subunit, Arg226, Leu233, and Met 234. By replacing each residue with Ala, we show that each contributes significantly to creating the R:C interface. By also deleting the second CNB domain (CNB-B), we show furthermore that CNB-B is a critical part of the cAMP-induced conformational switch that dislodges the B/C helix from the surface of the catalytic subunit. Without CNB-B the K{sub a} for activation by cAMP increases from 80 to 1000 nM. Replacing any of the key interface residues with Ala reduces the K{sub a} to 25-40 nM. Leu233 and M234 contribute to a hydrophobic latch that binds the B/C helix onto the large lobe of the C-subunit, while Arg226 is part of an electrostatic switch that couples the B/C helix to the phosphate binding cassette where the cAMP docks.

59 BASIC BIOLOGICAL SCIENCES↗