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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 325 records · Page 18
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Propane Dehydrogenation on Pt x Zn y Active Sites in Silicalite‐1

Abstract The improvement of Pt‐based catalysts for propane dehydrogenation (PDH) has progressed by recent investigations that have identified Zn as a promising promoter for Pt subnanometer catalysts. It is desirable to gain insights into the structure, stability, and activity of such active sites and the factors that influence them, such as Zn : Pt ratio, Pt coordination and nuclearity. Here, we employ density functional theory and microkinetic simulations to investigate the stability of Pt x Zn y ( x =1–3, y=0–3) active sites grafted on silanols of Silicalite‐1 and the PDH activity of Pt. We find that the coordination of a Pt atom to a nest of grafted Zn(II) atoms increases the stability of the Pt 1 Zn y sites, whose activity is similar for y=0–2 and drops dramatically for y>2. We further demonstrate, via linear scaling relations and microkinetic simulations, that the turnover frequency obeys a volcano law as a function of propylene binding strength. The Pt 2 Zn 1 and Pt 3 Zn 1 sites are stable and exhibit activity similar to Pt 1 Zn 2 , but only Pt 1 Zn 2 manifests reaction kinetics consistent with experimental data, strongly suggesting the active site composition in the synthesized catalyst samples. The methodology presented here suggests a general strategy for deducing active site information such as composition through simple kinetic experiments.

Liu, Yilang↗
DOE OSTI2021

Single‐Atomic Site Catalyst with Heme Enzymes‐Like Active Sites for Electrochemical Sensing of Hydrogen Peroxide

Abstract Heme enzymes, with the pentacoordinate heme iron active sites, possess high catalytic activity and selectivity in biosensing applications. However, they are still subject to limited catalytic stability in the complex environment and high cost for broad applications in electrochemical sensing. It is meaningful to develop a novel substitute that has a similar structure to some heme enzymes and mimics their enzyme activities. One emerging strategy is to design the Fe‐N‐C based single‐atomic site catalysts (SASCs). The obtained atomically dispersed Fe‐N x active sites can mimic the active sites of heme enzymes effectively. In this work, a SASC (Fe‐SASC/NW) is synthesized by doping single iron atoms in polypyrrole (PPy) derived carbon nanowire via a zinc‐atom‐assisted method. The proposed Fe‐SASC/NW shows high heme enzyme‐like catalytic performance for hydrogen peroxide (H 2 O 2 ) with a specific activity of 42.8 U mg −1 . An electrochemical sensor based on Fe‐SASC/NW is developed for the detection of H 2 O 2 . This sensor exhibits a wide detection concentration range from 5.0 × 10 −10 m to 0.5 m and an excellent limit of detection (LOD) of 46.35 × 10 −9 m . Such excellent catalytic activity and electrochemical sensing sensitivity are attributed to the isolated Fe‐N x active sites and their structural similarity with natural metalloproteases.

Ding, Shichao↗
DOE OSTI2023

Tailoring activity of iron phthalocyanine by edge-nitrogen sites induced electronic delocalization

Fe-N-C catalysts have been recognized as the most satisfactory candidates alternatively to Pt-based catalysts for oxygen reduction reaction (ORR). However, fine-tailoring of their intrinsic ORR catalytic activity still remains a great challenge due to the inferior accessibility and intrinsic activity of FeN x moieties. Herein, one order of magnitude activity enhancement of pristine Fe-N-C through cooperating with nitrogen-doped carbon micro-flower is achieved. The axial coordination effect between Fe active center and nitrogen atoms in support can break the electronic distribution symmetry of FeN x moieties and induce the electron delocalization on Fe active center and the electron localization on N, respectively, which favor the adsorption behavior of *OH intermediate. As a result, the catalyst exhibits a remarkable half-wave potential of 0.9 V and a high kinetic current density of 74.04 mA cm -2 at 0.85 V. In addition, when utilized as a cathode catalyst of liquid Zn-air batteries (ZABs), it possesses excellent electrochemical performance, for example, a high open circuit voltage (OCV) and peak power density of 1.59 V and 170.09 mW cm -2 , respectively. In conclusion, this work provides a new understanding into the activity enhancement mechanism of Fe-N-C catalysts, and inspires electronic delocalization of active sites for adjusting catalytic activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2026

Hierarchical semi-Markov models with duration-aware dynamics for activity sequences

Residential electricity demand at granular scales is driven by what people do and for how long. Accurately forecasting this demand for applications like microgrid management and demand response therefore requires generative models for activities that can produce realistic daily activity sequences, capturing both the timing and duration of human behavior. This paper develops a generative model of human activity sequences using nationally representative time-use diaries at a 10-min resolution. We use this model to quantify which demographic factors are most critical for improving predictive performance. We propose a hierarchical semi-Markov framework that addresses two key modeling challenges. First, a time-inhomogeneous Markov router learns the patterns of “which activity comes next.” Second, a semi-Markov hazard component explicitly models activity durations, capturing “how long” activities realistically last. To ensure statistical stability when data are sparse, the model pools information across related demographic groups and time blocks. The entire framework is trained and evaluated using survey design weights to ensure our findings are representative of the U.S. population. On a held-out test set, we demonstrate that explicitly modeling durations with the hazard component provides a substantial and statistically significant improvement over purely Markovian models. Furthermore, our analysis reveals a clear hierarchy of demographic factors: Sex, Day-Type, and Household Size provide the largest predictive gains, while Region and Season, though important for energy calculations, contribute little to predicting the activity sequence itself. The result is an interpretable and robust generator of synthetic activity traces, providing a high-fidelity foundation for downstream energy systems modeling.

24 POWER TRANSMISSION AND DISTRIBUTION↗
DOE OSTI2021

Indole-3-carbinol ameliorates necroptosis and inflammation of intestinal epithelial cells in mice with ulcerative colitis by activating aryl hydrocarbon receptor

Highlights: • AHR activated by I3C plays an important role in protecting IECs from necroptosis and inflammation, in vivo and in vitro. • AHR could ubiquitinate RIPK1, promote IAPs expression and inhibit NF-κB activation in NCM460 cells. • I3C is a good candidate as a natural product to prevent and treat UC. Ulcerative colitis (UC) is a disease characterized by inflammation and disruption of the intestinal epithelial barrier. Necroptosis plays a critical role in disease progression. Indole-3-carbinol (I3C), a natural dietary agonist of aryl hydrocarbon receptor (AHR), has shown alleviating effects on UC. However, its mechanisms of action have not been comprehensively elucidated. Therefore, we aimed at investigating the protective role of I3C in DSS-induced colitis mice models. I3C significantly ameliorated body weight loss, colon length shortening and colonic pathological damage in colitis mice, reduced disease activity index (DAI) and histological (HI) scores, as well as alleviated colonic necroptosis and inflammation. In vitro, I3C attenuated necroptosis and inflammation of colonoids and NCM460 cells. AHR, activated by I3C, inhibits activation of receptor-interacting protein kinase 1 (RIPK1) and the subsequent assembly of necrosome in a time-dependent manner, as well as suppressing NF-κB activation and decreasing TNF-α, IL-1β, IL-6 and IL-8 expression. Silencing of AHR aggravated necroptosis and inflammation of NCM460 cells, and did not be ameliorated by I3C. Furthermore, AHR activation induces the expression of inhibitor of apoptosis proteins (IAPs) and the ubiquitination of RIPK1. In conclusion, I3C exerts a protective effect in DSS-induced colitis mice models by alleviating the necroptosis and inflammation of IECs through activating AHR.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Ligand-Based Compound Activity Prediction via Few-Shot Learning

Predicting the activities of new compounds against biophysical or phenotypic assays based on the known activities of one or a few existing compounds is a common goal in early stage drug discovery. This problem can be cast as a “few-shot learning” challenge, and prior studies have developed few-shot learning methods to classify compounds as active versus inactive. However, the ability to go beyond classification and rank compounds by expected affinity is more valuable. We describe Few-Shot Compound Activity Prediction (FS-CAP), a novel neural architecture trained on a large bioactivity data set to predict compound activities against an assay outside the training set, based on only the activities of a few known compounds against the same assay. Our model aggregates encodings generated from the known compounds and their activities to capture assay information and uses a separate encoder for the new compound whose activity is to be predicted. The new method provides encouraging results relative to traditional chemical-similarity-based techniques as well as other state-of-the-art few-shot learning methods in tests on a variety of ligand-based drug discovery settings and data sets.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

CASSCF with Extremely Large Active Spaces Using the Adaptive Sampling Configuration Interaction Method

The complete active space self-consistent field (CASSCF) method is the principal approach employed for studying strongly correlated systems. However, exact CASSCF can only be performed on small active spaces of ~20 electrons in ~20 orbitals due to exponential growth in the computational cost. Here, we show that employing the Adaptive Sampling Configuration Interaction (ASCI) method as an approximate Full CI solver in the active space allows CASSCF-like calculations within chemical accuracy (<1 kcal/mol for relative energies) in active spaces with more than ~50 active electrons in ~50 active orbitals, significantly increasing the sizes of systems amenable to accurate multiconfigurational treatment. The main challenge with using any selected CI-based approximate CASSCF is the orbital optimization problem; they tend to exhibit large numbers of local minima in orbital space due to their lack of invariance to active–active rotations (in addition to the local minima that exist in exact CASSCF). We highlight methods that can avoid spurious local extrema as a practical solution to the orbital optimization problem. We employ ASCI-SCF to demonstrate a lack of polyradical character in moderately sized periacenes with up to 52 correlated electrons and compare against heat-bath CI on an iron porphyrin system with more than 40 correlated electrons.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Correlating Oxidation State and Surface Area to Activity from Operando Studies of Copper CO Electroreduction Catalysts in a Gas-Fed Device

Copyright © 2020 American Chemical Society. The rational design of high-performance electrocatalysts requires a detailed understanding of dynamic changes in catalyst properties, including oxidation states, surface area, and morphology under realistic working conditions. Oxide-derived Cu catalysts exhibit a remarkable selectivity toward multicarbon products for the electrochemical CO reduction reaction (CORR), but the exact role of the oxide remains elusive for explaining the performance enhancements. Here, we used operando X-ray absorption spectroscopy (XAS) coupled with simultaneous measurements of the catalyst activity and selectivity by gas chromatography (GC) to study the relationship between oxidation states of Cu-based catalysts and the activity for ethylene (C2H4) production in a CO gas-fed cell. By utilizing a custom-built XAS cell, oxidation states of Cu catalysts can be probed in device-relevant settings and under high current densities (>80 mA cm-2) for the CORR. By employing an electrochemical oxidation process, we found that the Cu oxidation states and specific ion species do not correlate with C2H4 production. The difference in the CORR activity is also investigated in relation to electrochemical surface area (ECSA) changes. While the hydrogen evolution reaction (HER) activity is positively correlated to the ECSA changes, the increased C2H4 activity is not proportional to the ECSA. Ex situ characterization from microscopic techniques suggests that the changes in the C2H4 activity and selectivity may arise from a morphological transformation that evolves into a more active structure. These comprehensive results give rise to the development of a cell regeneration method that can restore the performance of the Cu catalyst without cell disassembly. Our study establishes a basis for the rational design of highly active electrocatalysts for broad-range reactions in a gas-fed device.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Hydrogen Evolution on Restructured B-Rich WB: Metastable Surface States and Isolated Active Sites

Borides have been recently identified to be a class of earth-abundant and low-cost materials that are surprisingly active toward oxidative dehydrogenation and hydrogen evolution reaction (HER) catalysis. Here, we explain from first-principles calculations the HER activity of WB, an industrial material known for its superior physical properties and chemical inertness. We find that, compared to bulk termination, a major surface reconstruction takes place, leading to the off-stoichiometric B-rich surface termination that contains the active sites. The hydrogen adsorbate configurations are further investigated under various adsorbate coverages. Many competing configurations appear to be accessible in reaction conditions, and thus, a grand canonical ensemble is established to describe the catalytic system. A phase diagram of adsorbate coverages is constructed as a function of pH and the applied potential. A complex reaction network is presented based on the ensemble-averaged reaction rates, and the electrocatalytic activities are evaluated under different overpotentials. Here, the major contributors to the activity are found to be a few metastable surface states with a distinct electronic structure that are only accessible at high adsorbate coverages in reaction conditions. In addition, while the activity of the dominant active site is nearly the same as that on the unreconstructed WB, the B-rich formations play an important role of isolating the active sites and preventing the passivation of the surface with H 2 bubble formation.

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

Dynamically Formed Active Sites on Liquid Boron Oxide for Selective Oxidative Dehydrogenation of Propane

Boron-based catalysts have been shown to be both active and selective for driving the oxidative dehydrogenation of propane (ODHP) without the use of precious metals. This reaction occurs at temperatures that melt the oxide catalyst which challenges our ability to identify the liquid structures of the boron oxide phase under reaction conditions, hindering the understanding of its active sites and reaction mechanism. By combining ab initio molecular dynamics simulation, in-situ Raman characterization, and microkinetic modeling, we propose that the di-coordinated boron sites (BO2) in liquid boron oxide are the active species for O2 activation under reaction conditions. The formed peroxy-like species (>B-O-O-B<) can be viewed as a moderate oxidant for ODHP. The dynamical >B-O* dangling bond originated from >B-O-O-B< site as well as the liquid B2O3 structure itself, plays a critical role in the abstraction of H atoms from propane (C3H7 radical formation). Microkinetic modeling reveals C3H7 radical formation to be the main rate controlling step (~75% degree of rate control) with the dehydration of boron hydroxyls (B-OHs) to recover the di-coordinated boron active sites controlling the remainder of the rate (~25% degree of rate control). Moreover, the activation barriers are found to strongly depend upon the surface B-OH concentration. These findings provide significant insights into the active site and reaction mechanisms on boron-based catalysts for ODHP and underlie the importance of understanding the liquid nature of the catalyst to account for the catalytic activity.

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

The Enzymatic Activity of Inosine 5'-Monophosphate Dehydrogenase May Not Be a Vulnerable Target for Staphylococcus aureus Infections

Many bacterial pathogens, including Staphylococcus aureus, require inosine 5'-monophosphate dehydrogenase (IMPDH) for infection, making this enzyme a promising new target for antibiotics. Although potent selective inhibitors of bacterial IMPDHs have been reported, relatively few have displayed antibacterial activity. Here we use structure-informed design to obtain inhibitors of S. aureus IMPDH (SaIMPDH) that have potent antibacterial activity (minimal inhibitory concentrations less than 2 μM) and low cytotoxicity in mammalian cells. The physicochemical properties of the most active compounds were within typical Lipinski/Veber space, suggesting that polarity is not a general requirement for achieving antibacterial activity. In this work, five compounds failed to display activity in mouse models of septicemia and abscess infection. Inhibitor-resistant S. aureus strains readily emerged in vitro. Resistance resulted from substitutions in the cofactor/inhibitor binding site of SaIMPDH, confirming on-target antibacterial activity. These mutations decreased the binding of all inhibitors tested, but also decreased catalytic activity. Nonetheless, the resistant strains had comparable virulence to wild-type bacteria. Surprisingly, strains expressing catalytically inactive SaIMPDH displayed only a mild virulence defect. Collectively these observations question the vulnerability of the enzymatic activity of SaIMPDH as a target for the treatment of S. aureus infections, suggesting other functions of this protein may be responsible for its role in infection.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

A High-Entropy Oxide as High-Activity Electrocatalyst for Water Oxidation

High-entropy materials are an emerging pathway in the development of high-activity (electro)catalysts because of the inherent tunability and coexistence of multiple potential active sites, which may lead to earth-abundant catalyst materials for energy-efficient electrochemical energy storage. In this report, we identify how the multication composition in high-entropy perovskite oxides (HEO) contributes to high catalytic activity for the oxygen evolution reaction (OER), i.e., the key kinetically limiting half-reaction in several electrochemical energy conversion technologies, including green hydrogen generation. We compare the activity of the (001) facet of LaCr 0.2 Mn 0.2 Fe 0.2 Co 0.2 Ni 0.2 O 3-δ with the parent compounds (single B-site in the ABO 3 perovskite). While the single B-site perovskites roughly follow the expected volcano-type activity trends, the HEO clearly outperforms all of its parent compounds with 17 to 680 times higher currents at a fixed overpotential. As all samples were grown as an epitaxial layer, our results indicate an intrinsic composition–function relationship, avoiding the effects of complex geometries or unknown surface composition. In-depth X-ray photoemission studies reveal a synergistic effect of simultaneous oxidation and reduction of different transition metal cations during the adsorption of reaction intermediates. The surprisingly high OER activity demonstrates that HEOs are a highly attractive, earth-abundant material class for high-activity OER electrocatalysts, possibly allowing the activity to be fine-tuned beyond the scaling limits of mono- or bimetallic oxides.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Structural basis for Ca2+-dependent activation of a plant metacaspase

Abstract Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca 2+ for their activation and substrate processing. However, the Ca 2+ -dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana ( At MC4) that modulates Ca 2+ -dependent, damage-induced plant immune defense. The At MC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of At MC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca 2+ . Our analysis provides insight into the Ca 2+ -dependent activation of At MC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Structural basis of BAK activation in mitochondrial apoptosis initiation

BCL-2 proteins regulate mitochondrial poration in apoptosis initiation. How the pore-forming BCL-2 Effector BAK is activated remains incompletely understood mechanistically. Here we investigate autoactivation and direct activation by BH3-only proteins, which cooperate to lower BAK threshold in membrane poration and apoptosis initiation. We define in trans BAK autoactivation as the asymmetric “BH3-in-groove” triggering of dormant BAK by active BAK. BAK autoactivation is mechanistically similar to direct activation. The structure of autoactivated BAK BH3-BAK complex reveals the conformational changes leading to helix α1 destabilization, which is a hallmark of BAK activation. Helix α1 is destabilized and restabilized in structures of BAK engaged by rationally designed, high-affinity activating and inactivating BID-like BH3 ligands, respectively. Altogether our data support the long-standing hit-and-run mechanism of BAK activation by transient binding of BH3-only proteins, demonstrating that BH3-induced structural changes are more important in BAK activation than BH3 ligand affinity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Monitoring of asteroids in cometary orbits and activated asteroids through archival images and new observations

Transitional objects are minor bodies that share some characteristics with asteroids and others with comets. These objects include asteroids in cometary orbits (ACOs), which behave dynamically like comets, but lack observed activity, while activated asteroids (AAs) follow typical asteroidal orbits, but have shown dust ejections. The monitoring of a set of these objects carried out in 2015 and 2016 is continued using archival images from various observatories and new data from the IMPACTON telescope in Brazil. Two techniques were applied to detect activity: (i) surface brightness profiles were compared with those of field stars to identify widening, and (ii) the magnitudes reported in the Minor Planet Center, combined with our observations, were reduced and analyzed to identify abrupt brightness increases as a function of heliocentric distance. We analyzed the surface brightness profiles of 133 ACOs and 7 AAs. To study the reduced magnitude, we obtained data from the 705 ACOs that were known at the time of the analysis. Together with the data from our previous work, our analysis covered 23% of the total known ACOs; 8 deviated slightly in the surface brightness profile, 6 brightened in the reduced magnitude, and one object is in common in both samples. A very low percentage of objects might show activity (4% of the sample with brightness profiles and <1% in the reduced magnitudes). These results would rule out a slow transition from active to inert. Regarding AAs, 4 showed activity, and 3 of them matched previously reported periods, while the data we analyzed for P/2015 X 6 were obtained 19 days before the first existing activity report. The activity episodes of these objects are very restricted in time and do not always occur in the same region of the orbit.

asteroids: general↗
DOE OSTI2020

Revisiting heat treatment and surface activation of GaAs photocathodes: In situ studies using scanning tunneling microscopy and photoelectron spectroscopy

The lifetime of GaAs photocathodes in polarized electron guns is limited due to the delicate activation layer. An atomically clean and smooth GaAs surface is needed to deposit a robust activation layer, such as Cs 2 Te, with longer lifetime compared to traditional (Cs,O) activation. A previous experiment with Cs 2 Te activation on GaAs used heat cleaning temperatures around 400°C to avoid an increase in surface roughness [Bae et al., Appl. Phys. Lett. 112, 154101 (2018)]. Furthermore, high-temperature heat cleaning around 580°C, which results in a relatively contamination-free surface, could be one possible way to improve quantum efficiency. However, one should be cautious about surface roughness degradation during high-temperature heat cleaning. In this paper, we report results of surface roughness measurements on native, heat cleaned, and (Cs,O) activated GaAs photocathodes under vacuum. The results, measured by ultrahigh vacuum scanning tunneling microscopy, show that the surface roughness improves as the heat cleaning temperature is increased, by at least a factor of three for 580°C heat cleaning, compared to the native sample. Activation with (Cs,O) is shown to increase surface roughness by a factor of four compared to a 580°C heat cleaned sample. This confirms that high-temperature heat cleaning can be useful for depositing good quality robust activation layers on GaAs. Additionally, we also report chemical analysis for each step of preparation for p-doped GaAs photocathodes using X-ray photoelectron spectroscopy (XPS), angle-resolved XPS, and ultraviolet photoelectron spectroscopy. Our results indicate that the (Cs,O) activation layer forms a sandwich structure consists of Cs and oxygen. We found no formation of any specific compound such as Cs 2 O or Cs 11 O 3 .

43 PARTICLE ACCELERATORS↗
DOE OSTI2025

Achieving designed texture and flows in bulk active nematics using optimal control theory

Being intrinsically nonequilibrium, active materials can potentially perform functions that would be thermodynamically forbidden in passive materials. However, active systems have diverse local attractors that correspond to distinct dynamical states, many of which exhibit chaotic turbulent-like dynamics and thus cannot perform work or useful functions. Designing such a system to choose a specific dynamical state is a formidable challenge. Motivated by recent advances enabling optogenetic control of experimental active materials, we describe an optimal control theory framework that identifies a spatiotemporal sequence of light-generated activity that drives an active nematic system toward a prescribed dynamical steady state. Active nematics are unstable to spontaneous defect proliferation and chaotic streaming dynamics in the absence of control. We demonstrate that optimal control theory can compute activity fields that redirect the dynamics into a variety of alternative dynamical programs and functions. This includes dynamically reconfiguring between states, selecting and stabilizing emergent behaviors that do not correspond to attractors, and are hence unstable in the uncontrolled system. Furthermore, our results provide a roadmap to leverage optical control methods to rationally design structure, dynamics, and function in a wide variety of active materials.

Complex systems theory↗
DOE OSTI2025

An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state

The RAF kinases (ARAF, BRAF, and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-3 2 :MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

BRAF↗