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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 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Changes within the central stalk of E. coli F 1 F o ATP synthase observed after addition of ATP

F 1 F o ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the F o motor that is transferred to the F 1 motor to catalyze ATP production, with flexible F 1 /F o coupling required for efficient catalysis. F 1 F o ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F 1 F o ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the F ο motor. This highlights the importance of the central rotor for flexible coupling of the F 1 and F o motors and provides further insight into the regulatory mechanism mediated by subunit ε.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular basis for cooperative binding and synergy of ATP-site and allosteric EGFR inhibitors

Abstract Lung cancer is frequently caused by activating mutations in the epidermal growth factor receptor (EGFR). Allosteric EGFR inhibitors offer promise as the next generation of therapeutics, as they are unaffected by common ATP-site resistance mutations and synergize with the drug osimertinib. Here, we examine combinations of ATP-competitive and allosteric inhibitors to better understand the molecular basis for synergy. We identify a subset of irreversible EGFR inhibitors that display positive binding cooperativity and synergy with the allosteric inhibitor JBJ-04-125-02 in several EGFR variants. Structural analysis of these complexes reveals conformational changes occur mainly in the phosphate-binding loop (P-loop). Mutation of F723 in the P-loop reduces cooperative binding and synergy, supporting a mechanism in which F723-mediated contacts between the P-loop and the allosteric inhibitor are critical for synergy. These structural and mechanistic insights will aid in the identification and development of additional inhibitor combinations with potential clinical value.

Beyett, Tyler S. (ORCID:0000000155097004)↗
DOE OSTI2022

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Structure of an open K ATP channel reveals tandem PIP 2 binding sites mediating the Kir6.2 and SUR1 regulatory interface

ATP-sensitive potassium (K ATP ) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. K ATP channel opening is stimulated by PIP 2 and inhibited by ATP. Mutations that increase channel opening by PIP 2 reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP 2 in K ATP channel function, previously solved open-channel structures have lacked bound PIP 2 , and mechanisms by which PIP 2 regulates K ATP channels remain unresolved. Here, we report the cryoEM structure of a K ATP channel harboring the neonatal diabetes mutation Kir6.2-Q52R, in the open conformation, bound to amphipathic molecules consistent with natural C18:0/C20:4 long-chain PI(4,5)P 2 at two adjacent binding sites between SUR1 and Kir6.2. The canonical PIP 2 binding site is conserved among PIP 2 -gated Kir channels. The non-canonical PIP 2 binding site forms at the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP 2 binding and gating, explain the antagonistic regulation of K ATP channels by PIP 2 and ATP, and provide a putative mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.

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

Conserved role of ATP synthase in mammalian cilia

Highlights: • ATP synthase localizes to cilia in mammalian cells. • ATP synthase is involved in ciliogenesis. • ATP synthase probably participates in polycystin signalling pathway. We previously found that ATP synthases localize to male-specific sensory cilia and control the ciliary response by regulating polycystin signalling in Caenorhabditis elegans. Herein, we discovered that the ciliary localization of ATP synthase is evolutionarily conserved in mammals. We showed that the ATP synthase subunit F1β is colocalized with the cilia marker acetylated α-tubulin in both mammalian renal epithelial cells (MDCK) and normal mouse cholangiocytes (NMCs). Treatment with ATP synthase inhibitor oligomycin impaired ciliogenesis in MDCK cells, and F1β was co-immunoprecipitated with PKD2 in mammalian cells. Our study provides evidence for the evolutionarily conserved localization of ATP synthase in cilia from worm to mammals. Defects in ATP synthase can lead to ciliary dysfunction, which may be a potential mechanism of polycystic kidney disease.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction

Adenosine-5’-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics’ impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses

Abstract Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana , Nicotiana tabacum , and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Vascular K ATP channel structural dynamics reveal regulatory mechanism by Mg-nucleotides

Vascular tone is dependent on smooth muscle K ATP channels comprising pore-forming Kir6.1 and regulatory SUR2B subunits, in which mutations cause Cantú syndrome. Unique among K ATP isoforms, they lack spontaneous activity and require Mg-nucleotides for activation. Structural mechanisms underlying these properties are unknown. Here, we determined cryogenic electron microscopy structures of vascular K ATP channels bound to inhibitory ATP and glibenclamide, which differ informatively from similarly determined pancreatic K ATP channel isoform (Kir6.2/SUR1). Unlike SUR1, SUR2B subunits adopt distinct rotational “propeller” and “quatrefoil” geometries surrounding their Kir6.1 core. The glutamate/aspartate-rich linker connecting the two halves of the SUR-ABC core is observed in a quatrefoil-like conformation. Molecular dynamics simulations reveal MgADP-dependent dynamic tripartite interactions between this linker, SUR2B, and Kir6.1. The structures captured implicate a progression of intermediate states between MgADP-free inactivated, and MgADP-bound activated conformations wherein the glutamate/aspartate-rich linker participates as mobile autoinhibitory domain, suggesting a conformational pathway toward K ATP channel activation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

CryoEM reveals the stochastic nature of individual ATP binding events in a group II chaperonin

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Expanding the use of ethanol as a feedstock for cell-free synthetic biochemistry by implementing acetyl-CoA and ATP generating pathways

Abstract Ethanol is a widely available carbon compound that can be increasingly produced with a net negative carbon balance. Carbon-negative ethanol might therefore provide a feedstock for building a wider range of sustainable chemicals. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochemicals, and how we can use the energy stored in ethanol to generate ATP, another key molecule important for powering biochemical pathways. The ATP generator produces acetone as a value-added side product. Our ATP generator reached titers of 27 ± 6 mM ATP and 59 ± 15 mM acetone with maximum ATP synthesis rate of 2.8 ± 0.6 mM/h and acetone of 7.8 ± 0.8 mM/h. We illustrated how the ATP generating module can power cell-free biochemical pathways by converting mevalonate into isoprenol at a titer of 12.5 ± 0.8 mM and a maximum productivity of 1.0 ± 0.05 mM/h. These proof-of-principle demonstrations may ultimately find their way to the manufacture of diverse chemicals from ethanol and other simple carbon compounds.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The role of photorespiration in preventing feedback regulation via ATP synthase in Nicotiana tabacum

Abstract Photorespiration consumes substantial amounts of energy in the forms of adenosine triphosphate (ATP) and reductant making the pathway an important component in leaf energetics. Because of this high reductant demand, photorespiration is proposed to act as a photoprotective electron sink. However, photorespiration consumes more ATP relative to reductant than the C3 cycle meaning increased flux disproportionally increases ATP demand relative to reductant. Here we explore how energetic consumption from photorespiration impacts the flexibility of the light reactions in nicotiana tabacum . Specifically, we demonstrate that decreased photosynthetic efficiency (ϕ II ) at low photorespiratory flux was related to feedback regulation at the chloroplast ATP synthase. Additionally, decreased ϕ II at high photorespiratory flux resulted in the accumulation of photoinhibition at photosystem II centers. These results are contrary to the proposed role of photorespiration as a photoprotective electron sink. Instead, our results suggest a novel role of ATP consumption from photorespiration in maintaining ATP synthase activity, with implications for maintaining energy balance and preventing photodamage that will be critical for plant engineering strategies.

Plant Sciences↗
DOE OSTI2023

Metallurgical and mechanical characterization of ATP-5 aluminum alloys for minimizing machining distortion

ATP-5 aluminum alloy was considered as an alternative to 6061-T651 aluminum alloy to minimize machining distortion. ATP-5 is a proprietary cast alloy that is compositionally similar to 5083 aluminum and is purported to have excellent machinability, stability, and corrosion response. Dimensional stability tests, mechanical testing, chemical analysis, microstructural analysis, and fractography were completed to understand the metallurgy of the ATP-5 alloy and assess its potential as a 6061-T651 alloy alternative. Ultimately, the ATP-5 was found to retain dimensional stability issues, albeit to a lesser extent, relative to the 6061-T651 alloy. Combined with the residual cast structure observed microstructurally and lot-to-lot mechanical property variation, the alloy was deemed not suitable for a structural application. Alternative uses of ATP-5 include tooling, molds, vacuum chucks, fixtures, and jigs, where the material can shine in non-load bearing applications with a need for dimensional control and machinability.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Transforming an ATP-dependent enzyme into a dissipative, self-assembling system

Nucleoside triphosphate (NTP)-dependent protein assemblies such as microtubules and actin filaments have inspired the development of diverse chemically fueled molecular machines and active materials but their functional sophistication has yet to be matched by design. Given this challenge, we asked whether it is possible to transform a natural adenosine 5'-triphosphate (ATP)-dependent enzyme into a dissipative self-assembling system, thereby altering the structural and functional mode in which chemical energy is used. Furthermore we report that FtsH (filamentous temperature-sensitive protease H), a hexameric ATPase involved in membrane protein degradation, can be readily engineered to form one-dimensional helical nanotubes. FtsH nanotubes require constant energy input to maintain their integrity and degrade over time with the concomitant hydrolysis of ATP, analogous to natural NTP-dependent cytoskeletal assemblies. Yet, in contrast to natural dissipative systems, ATP hydrolysis is catalyzed by free FtsH protomers and FtsH nanotubes serve to conserve ATP, leading to transient assemblies whose lifetimes can be tuned from days to minutes through the inclusion of external ATPases in solution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Structure of VanS from vancomycin-resistant enterococci: A sensor kinase with weak ATP binding

The VanRS two-component system regulates the resistance phenotype of vancomycin-resistant enterococci. VanS is a sensor histidine kinase that responds to the presence of vancomycin by autophosphorylating and subsequently transferring the phosphoryl group to the response regulator, VanR. The phosphotransfer activates VanR as a transcription factor, which initiates the expression of resistance genes. Structural information about VanS proteins has remained elusive, hindering the molecular-level understanding of their function. Here, we present X-ray crystal structures for the catalytic and ATP-binding (CA) domains of two VanS proteins, derived from vancomycin-resistant enterococci types A and C. Both proteins adopt the canonical Bergerat fold that has been observed for CA domains of other prokaryotic histidine kinases. We attempted to determine structures for the nucleotide-bound forms of both proteins; however, despite repeated efforts, these forms could not be crystallized, prompting us to measure the proteins' binding affinities for ATP. Unexpectedly, both CA domains displayed low affinities for the nucleotide, with K D values in the low millimolar range. Since these K D values are comparable to intracellular ATP concentrations, this weak substrate binding could reflect a way of regulating expression of the resistance phenotype.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

ATP Drives Efficient Terpene Biosynthesis in Marine Thraustochytrids

Understanding carbon flux controlling mechanisms in a tangled metabolic network is an essential question of cell metabolism. Secondary metabolism, such as terpene biosynthesis, has evolved with low carbon flux due to inherent pathway constraints. Thraustochytrids are a group of heterotrophic marine unicellular protists and can accumulate terpenoids under the high-salt conditions in their natural environment. However, the mechanism behind terpene accumulation is not well understood. Here, we show that terpene biosynthesis in Thraustochytrium sp. ATCC 26185 is constrained by local thermodynamics in the mevalonate pathway. Thermodynamic analysis reveals metabolite limitation in the nondecarboxylative Claisen condensation of acetyl-coenzyme A (CoA) to the acetoacetyl-CoA step, catalyzed by the acetyl-CoA acetyltransferase (ACAT). Through a sodium-elicited mechanism, higher respiration leads to increased ATP investment into the mevalonate pathway, providing a strong thermodynamic driving force for enhanced terpene biosynthesis. Proteomic and metabolomic analyses further show that the increased ATP demands are fulfilled by shifting energy generation from carbohydrate to lipid oxidation. This study demonstrates a unique strategy in nature that uses ATP to drive a low-flux metabolic pathway, providing an alternative solution for efficient terpene metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Effect of plasma treatment on LMPAEK/CF tape and composites manufactured by automated tape placement (ATP)

Automated tape placement (ATP) process is widely used in aerospace for its advanced process control and multi-axis capabilities but faces issues like limited choice of materials and suboptimal tape consolidation. This study investigates air plasma treatment on ATP carbon fiber thermoplastic feedstock tape to address these challenges. The effects on low melt Polyaryletherketone/carbon fiber unidirectional tape (LMPAEK/CF UD tape) were analyzed. Treated and untreated tapes were used to fabricate composites and evaluated for physical, thermal, mechanical, and interfacial properties. Atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) analyses revealed surface roughness changes (on LMPAEK), extent of oxidation, and the presence of hydroxyl/carboxyl groups. Composites from plasma-treated tapes showed a 7.6% increase in tensile strength, 8% in tensile modulus, 18% in flexural strength, and 8.3% in flexural modulus. Further, the interlaminar shear strength improved by 18.7%. Failure analysis showed untreated composites failed via inter-ply and fiber-matrix delamination, while treated composites experienced matrix cracking and fiber breakage. This study highlights atmospheric plasma treatment as a solution to ATP’s limitations, significantly enhancing LMPAEK/CF UD tape composites’ properties.

36 MATERIALS SCIENCE↗