Search NASA⌕ Search

SEARCH · Search NASA

Results for “Envelope analysis”

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.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 217 records · Page 12
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Mutations that confer resistance to broadly-neutralizing antibodies define HIV-1 variants of transmitting mothers from that of non-transmitting mothers

Despite considerable reduction of mother-to-child transmission (MTCT) of HIV through use of maternal and infant antiretroviral therapy (ART), over 150,000 infants continue to become infected with HIV annually, falling far short of the World Health Organization goal of reaching <20,000 annual pediatric HIV cases worldwide by 2020. Prior to the widespread use of ART in the setting of pregnancy, over half of infants born to HIV-infected mothers were protected against HIV acquisition. Yet, the role of maternal immune factors in this protection against vertical transmission is still unclear, hampering the development of synergistic strategies to further reduce MTCT. It has been established that infant transmitted/founder (T/F) viruses are often resistant to maternal plasma, yet it is unknown if the neutralization resistance profile of circulating viruses predicts the maternal risk of transmission to her infant. In this study, we amplified HIV-1 envelope genes ( env ) by single genome amplification and produced representative Env variants from plasma of 19 non-transmitting mothers from the U.S. Women Infant Transmission Study (WITS), enrolled in the pre-ART era. Maternal HIV Env variants from non-transmitting mothers had similar sensitivity to autologous plasma as observed for non-transmitting variants from transmitting mothers. In contrast, infant variants were on average 30% less sensitive to paired plasma neutralization compared to non-transmitted maternal variants from both transmitting and non-transmitting mothers (p = 0.015). Importantly, a signature sequence analysis revealed that motifs enriched in env sequences from transmitting mothers were associated with broadly neutralizing antibody (bnAb) resistance. Altogether, our findings suggest that circulating maternal virus resistance to bnAb-mediated neutralization, but not autologous plasma neutralization, near the time of delivery, predicts increased MTCT risk. These results caution that enhancement of maternal plasma neutralization through passive or active vaccination during pregnancy may potentially drive the evolution of variants fit for vertical transmission.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

The GAPS programme at TNG

Neptunes represent one of the main types of exoplanets and have chemical-physical characteristics halfway between rocky and gas giant planets. Therefore, their characterization is important for understanding and constraining both the formation mechanisms and the evolution patterns of planets. We investigate the exoplanet candidate TOI-1422 b, which was discovered by the TESS space telescope around the high proper-motion G2 V star TOI-1422 (V = 10.6 mag), 155 pc away, with the primary goal of confirming its planetary nature and characterising its properties. We monitored TOI-1422 with the HARPS-N spectrograph for 1.5 yr to precisely quantify its radial velocity (RV) variation. We analyse these RV measurements jointly with TESS photometry and check for blended companions through high-spatial resolution images using the AstraLux instrument. We estimate that the parent star has a radius of R$_\star$ = 1.019 -0.013 +0.014 R ⊙ , and a mass of M$_\star$ = 1.019 -0.013 +0.014 M ⊙ . Our analysis confirms the planetary nature of TOI-1422 b and also suggests the presence of a Neptune-mass planet on a more distant orbit, the candidate TOI-1422 c, which is not detected in TESS light curves. The inner planet, TOI-1422 b, orbits on a period of P b = 12.9972 ± 0.0006 days and has an equilibrium temperature of T eq,b = 867 ± 17 K. With a radius of R b = 3.96 -0.11 +0.13 R ⊕ , a mass of M b = 9.0 -2.0 +2.3 M ⊕ and, consequently, a density of ρ b = 0.795 -0.235 +0.290 g cm -3 , it can be considered a warm Neptune-sized planet. Compared to other exoplanets of a similar mass range, TOI-1422 b is among the most inflated, and we expect this planet to have an extensive gaseous envelope that surrounds a core with a mass fraction around 10% – 25% of the total mass of the planet. The outer non-transiting planet candidate, TOI-1422 c, has an orbital period of P c = 29.29 -0.20 +0.21 days, a minimum mass, M c sin i, of 11.1 -2.3 +2.6 M ⊕ , an equilibrium temperature of T eq,c = 661 ± 13 K and, therefore, if confirmed, could be considered as another warm Neptune.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2024

Methyl α‐D‐galactopyranosyl‐(1→3)‐β‐D‐galactopyranoside and methyl β‐D‐galactopyranosyl‐(1→3)‐β‐D‐galactopyranoside: Glycosidic linkage conformation determined from MA'AT analysis

Abstract MA'ATanalysis has been applied to two biologically‐importantO‐glycosidic linkages in two disaccharides, α‐D‐Galp‐(1→3)‐β‐D‐GalpOMe (3) and β‐D‐Galp‐(1→3)‐β‐D‐GalpOMe (4). Using density functional theory (DFT) to obtain parameterized equations relating a group of trans‐O‐glycosidic NMR spin‐couplings to eitherphi(ϕ') orpsi(ψ'), and experimental 3 J COCH , 2 J COC , and 3 J COCC spin‐couplings measured in aqueous solution in 13 C‐labeled isotopomers, probability distributions ofϕ'andψ'in each linkage were determined and compared to those determined by aqueous 1‐μs molecular dynamics (MD) simulation. Good agreement was found between theMA'ATand single‐state MD conformational models of these linkages for the most part, with modest (approximately <15°) differences in the mean values ofϕ'andψ', although the envelope of allowed angles (encoded in circular standard deviations or CSDs) is consistently larger forϕ'determined fromMA'ATanalysis than from MD for both linkages. TheMA'ATmodel of the α‐Galp‐(1→3)‐β‐Galplinkage agrees well with those determined previously using conventional NMR methods ( 3 J COCH values and/or 1 H‐ 1 H NOEs), but some discrepancy was observed for the β‐Galp‐(1→3)‐β‐Galplinkage, which may arise from errors in the conventions used to describe the linkage torsion angles. Statistical analyses of X‐ray crystal structures show ranges ofϕ'andψ'for both linkages that include the mean angles determined fromMA'ATanalyses, although both angles adopt a wide range of values in the crystalline state, withϕ'in β‐Galp‐(1→3)‐β‐Galplinkages showing greater‐than‐expected conformational variability.

Chemistry↗
DOE OSTI2022

Effective properties of semitransparent radiative cooling materials with spectrally variable properties

Radiative cooling is a promising solid-state, non-vapor-compression technology for passive refrigeration and air conditioning. Although this phenomenon occurs naturally, achieving a significant amount of cooling to make it a technically and economically viable technology requires highly engineered, spectrally selective radiative surfaces. These characteristics make radiative cooling difficult to estimate, particularly when it is integrated with other systems such as photovoltaic panels or building envelopes. The complexity further increases when the substrate also participates in the radiative cooling (along with the radiative coating). Energy estimation is becoming increasingly critical because of the recent focus on the semitransparent radiative coatings that transmit a variety of colors to enhance the aesthetic appeal of the system. In this work, we propose an iterative method to calculate the effective radiative properties, which provides the same net radiative cooling that would be observed using the spectral properties at both the coating and substrate surfaces. Compared to traditional methods that rely on either computationally expensive full spectral analysis or methods for averaging each radiative surface parameter locally, our proposed method focuses on calculating effective properties that provide the same the net cooling effect as a full spectral analysis by accounting the emissivity, absorptivity, and transmissivity collectively, thereby providing an overall estimation error of less than 0.2%. We believe that this study will be beneficial to the engineering communities that employ complex simulation codes and require lumped solar and thermal radiation related parameters.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2020

The host galaxies of 106 rapidly evolving transients discovered by the Dark Energy Survey

Rapidly evolving transients (RETs), also termed fast blue optical transients, are a recently discovered group of astrophysical events that display rapid luminosity evolution. RETs typically rise to peak in less than 10 d and fade within 30, a time-scale unlikely to be compatible with the decay of Nickel-56 that drives conventional supernovae (SNe). Their peak luminosity spans a range of -15 < Mg < -22.5, with some events observed at redshifts greater than 1. Their evolution on fast time-scales has hindered high-quality follow-up observations, and thus their origin and explosion/emission mechanism remains unexplained. In this paper, we present the largest sample of RETs to date, comprising 106 objects discovered by the Dark Energy Survey, and perform the most comprehensive analysis of RET host galaxies. Using deep-stacked photometry and emission lines from OzDES spectroscopy, we derive stellar masses and star formation rates (SFRs) for 49 host galaxies, and metallicities ([O/H]) for 37. Here, we find that RETs explode exclusively in star-forming galaxies and are thus likely associated with massive stars. Comparing RET hosts to samples of host galaxies of other explosive transients as well as field galaxies, we find that RETs prefer galaxies with high specific SFRs ($\langle$log(sSFR)$\rangle$ ~ -9.6), indicating a link to young stellar populations, similar to stripped-envelope SNe. RET hosts appear to show a lack of chemical enrichment, their metallicities akin to long-duration gamma-ray bursts and superluminous SN host galaxies ($\langle$12+ log (O/H)$\rangle$ ~ 9.4). There are no clear relationships between mass or SFR of the host galaxies and the peak magnitudes or decline rates of the transients themselves.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2024

Integrated Off-Gas System Tests on the DM1200 Melter with RPP-WTP LAW Sub-Envelope Bl Simulants, VSL-03R3851-1, Rev. 0 (Oct 2003)

The report documents melter and off-gas performance results obtained on the DM1200 melter during processing of LAW Sub-Envelope B1 feed. The principal objectives of the DM1200 melter testing were to demonstrate suitable processing and production rates for LAW B1 feed; characterize the glass product for elemental composition; measure elemental emission rates and decontamination factors (DFs) across the melter and off-gas treatment components; and provide additional data on the performance of the thermal catalytic oxidation and selective catalytic reduction (TCO/SCR) unit efficiency in treating organic emissions. The feed was spiked with organic compounds for part of the testing duration. All data were collected for use in engineering and system design purposes. No sampling and analysis to collect environmental data was performed during these tests.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2022

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

Abstract The intrinsic tenase (Xase) complex, formed by factors (f) VIIIa and fIXa, forms on activated platelet surfaces and catalyzes the activation of factor X to Xa, stimulating thrombin production in the blood coagulation cascade. The structural organization of the membrane-bound Xase complex remains largely unknown, hindering our understanding of the structural underpinnings that guide Xase complex assembly. Here, we aimed to characterize the Xase complex bound to a lipid nanodisc with biolayer interferometry (BLI), Michaelis–Menten kinetics, and small-angle X-ray scattering (SAXS). Using immobilized lipid nanodiscs, we measured binding rates and nanomolar affinities for fVIIIa, fIXa, and the Xase complex. Enzyme kinetic measurements demonstrated the assembly of an active enzyme complex in the presence of lipid nanodiscs. An ab initio molecular envelope of the nanodisc-bound Xase complex allowed us to computationally model fVIIIa and fIXa docked onto a flexible lipid membrane and identify protein–protein interactions. Our results highlight multiple points of contact between fVIIIa and fIXa, including a novel interaction with fIXa at the fVIIIa A1–A3 domain interface. Lastly, we identified hemophilia A/B-related mutations with varying severities at the fVIIIa/fIXa interface that may regulate Xase complex assembly. Together, our results support the use of SAXS as an emergent tool to investigate the membrane-bound Xase complex and illustrate how mutations at the fVIIIa/fIXa dimer interface may disrupt or stabilize the activated enzyme complex.

Hematology↗
DOE OSTI2020

The shape of SN 1993J re-analysed

SN 1993J is one of the best-studied Type IIb supernovae. Spectropolarimetric data analyses were published over two decades ago at a time when the field of supernova spectropolarimetry was in its infancy. Here, we present a new analysis of the spectropolarimetric data of SN 1993J and an improved estimate of its interstellar polarization (ISP) as well as a critical review of ISP removal techniques employed in the field. The polarization of SN 1993J is found to show significant alignment on the q - u plane, suggesting the presence of a dominant axis and therefore of continuum polarization. We also see strong line polarization features, including H β, He i λ5876, H α, He i λ6678, He i λ7065, and high velocity (HV) components of He i λ5876 and H α. SN 1993J is therefore the second example of a stripped-envelope supernova, alongside iPTF13bvn, with prominent HV helium polarization features, and the first to show a likely HV H α contribution. Overall, we determine that the observed features can be interpreted as the superposition of anisotropically distributed line forming regions over ellipsoidal ejecta. We cannot exclude the possibility of an off-axis energy source within the ejecta. These data demonstrate the rich structures that are inaccessible if solely considering the flux spectra but can be probed by spectropolarimetric observations. In future studies, the new ISP corrected data can be used in conjunction with 3D radiative transfer models to better map the geometry of the ejecta of SN 1993J.

79 ASTRONOMY AND ASTROPHYSICS↗
DOE OSTI2026

Ceramic Composite Experimental Testing Status

Over recent years, ceramic matrix materials such as SiC–SiC and C–C have been gaining interest for use in fusion reactors, light water reactors (LWRs), and high-temperature reactors (HTRs). These materials are good candidates to operate in very high temperature and moderate to high radiation environments. The evaluation of composite materials, in general, is challenging because of variations in precursor materials, variations in the fabrication process across fabricators, and the wide range of potential fiber architectures, to name a few. However, the need to evaluate neutron-irradiated properties adds another layer of complexity, which includes cost, timeline, and specimen size limitations (often associated with irradiation testing). A qualification methodology for the use of ceramic composites is provided in the American Society of Mechanical Engineers (ASME) Boiler and Pressure Vessel Code Section III-5-HHB. The methodology is supported by ASTM International (ASTM) guides, which provide a pathway to accomplish this effort. Part of the qualification strategy is for the designer to collect material property data on environmental conditions representative of its design envelope. These data include irradiation effects. This report presents an experimental study and test campaign developed to partially address this gap by providing initial mechanical and physical property data required for design. A variety of different materials using different manufacturing techniques are considered as part of this campaign. The test plan suggests performing a screening or partial irradiation study to assist the designer during the material selection process. The designer can then perform a more comprehensive qualification study if the material performance is promising. This work focuses on the status of the specimen preparations (machining of samples), the current test methods and failure analysis as well as the preparation of irradiation vehicles for the irradiation campaign. The irradiation will be performed at Oak Ridge National Laboratory (ORNL) in the High Flux Isotope Reactor (HFIR) and at Idaho National Laboratory (INL) in the Advanced Test Reactor (ATR).

22 GENERAL STUDIES OF NUCLEAR REACTORS↗
DOE OSTI2019

Modulational instability and soliton trains in a model for two-mode fiber ring lasers

A model of orthogonally polarized two-field fiber ring laser with a linear gain is considered, with emphasis on the continuous-wave stability and the existence of soliton trains. The continuous-wave stability analysis is carried out within the framework of the modulational-instability approach, the variations of the gain spectrum with the modulation frequency and characteristic parameters of the model give rise to a rich variety of stability features including single-band and multiband stability regions. Seeking for pulse structures of the model, the two coupled cubic complex Ginzburg–Landau equations describing individual mode propagations are transformed into a set of coupled, first-order nonlinear ordinary-differential equations for the amplitudes and phases of the two modes. Numerical simulations of the last set of coupled equations indicate that in the anomalous dispersion regime, envelopes of the two fields are periodic trains of pulses the amplitudes of which are affected by the linear gain.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2020

A low-luminosity core-collapse supernova very similar to SN 2005cs

ABSTRACT We present observations and analysis of PSN J17292918+7542390, a low-luminosity Type II-P supernova (LL SN IIP). The observed sample of such events is still low, and their nature is still under debate. Such SNe are similar to SN 2005cs, a well-observed LL Type II-P event, having low expansion velocities, and small ejected 56Ni mass. We have developed a robust and relatively fast Monte Carlo code that fits semi-analytic models to light curves of core-collapse SNe. This allows the estimation of the most important physical parameters, like the radius of the progenitor star, the mass of the ejected envelope, the mass of the radioactive nickel synthesized during the explosion, among others. PSN J17292918+7542390 has $R_0 = 91_{-70}^{+119} \times 10^{11} \, \text{cm}$, $M_\text{ej} = 9.89_{-1.00}^{+2.10} \, \mathrm{ M}_{\odot }$, $E_{\mbox{kin}} = 0.65_{-0.18}^{+0.19} \, \text{foe}$, and $v_{\mbox{exp}} = 3332_{-347}^{+216}$ km s−1, for its progenitor radius, ejecta mass, kinetic energy, and expansion velocity, respectively. The initial nickel mass of the PSN J17292918+7542390 turned out to be $1.55_{-0.70}^{+0.75} \times 10^{-3} \, \mathrm{M}_{\odot }$. The measured photospheric velocity at the earliest observed phase is 7000 km s−1. As far as we can tell based on the small population of observed LL SNe IIP, the determined values are typical for these events.

Jäger, Jr, Zoltán↗
DOE OSTI2020

Residential Natural Gas Demand Response Potential during Extreme Cold Events in Electricity-Gas Coupled Energy Systems

In regions where natural gas is used for both power generation and heating buildings, extreme cold weather events can place the electrical system under enormous stress and challenge the ability to meet residential heating and electric demands. Residential demand response has long been used in the power sector to curtail summer electric load, but these types of programs in general have not seen adoption in the natural gas sector during winter months. Natural gas demand response (NG-DR) has garnered interest given recent extreme cold weather events in the United States; however, the magnitude of savings and potential impacts—to occupants and energy markets—are not well understood. We present a case-study analysis of the technical potential for residential natural gas demand response in the northeast United States that utilizes diverse whole-building energy simulations and high-performance computing. Our results show that NG-DR applied to residential heating systems during extreme cold-weather conditions could reduce natural gas demand by 1–29% based on conservative and aggressive strategies, respectively. This indicates a potential to improve the resilience of gas and electric systems during stressful events, which we examine by estimating the impact on energy costs and electricity generation from natural gas. We also explore relationships between hourly indoor temperatures, demand response, and building envelope efficiency.

03 NATURAL GAS↗
DOE OSTI2023

Probing the in situ volumes of Arabidopsis leaf plastids using three‐dimensional confocal and scanning electron microscopy

SUMMARY Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block‐face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild‐type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col‐0 wild‐type mesophyll chloroplasts is 93 μm 3 . Wild‐type guard cell plastids are approximately 18 μm 3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

Knoblauch, Jan↗
DOE OSTI2023

Building Science Education for Solar Decathlon: Emissions and the Built Environment [Slides]

The Solar Decathlon Building Science Education series is designed to educate students and working professionals on building science principles that are paramount to the successful design of high-performance, energy-efficient buildings. Instructional content is presented in modules, covering specific topics. Altogether, this series aims to educate viewers on: 1) where/how energy is used in buildings; 2) how to define zero energy buildings; 3) how to apply the fundamentals of thermodynamics to building envelope design; 4) how to explain the science of how/why buildings use energy; 5) how to apply this knowledge to design comfortable energy efficient buildings. Students and working professionals can use this educational information at no cost to complement academic curriculum and continuing education activities. This is Module 7 which focuses on embodied environmental carbon.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2024

Emulation and detection of physical faults and cyber-attacks on building energy systems through real-time hardware-in-the-loop experiments

The increasing use of remote or mobile access, integrated wearable technologies, data exchange, and cloud-based data analytics in modern smart buildings is steering the building industry towards open communication technologies. The increased connectivity and accessibility could lead to more cyber-attacks in smart buildings. On the other hand, physical faults (e.g., HVAC -heating, ventilation, and air-conditioning faults) may have similar adverse impacts as those from the cyber-attacks on building energy systems, such as occupant discomfort, energy wastage, and equipment downtime. However, current physical behavior-based anomaly detection methods fail to differentiate between cyber-attacks and physical faults in building energy systems. Moreover, the challenge in collecting real-world threat data with ground truth has led researchers to rely on numerical models with user-defined assumptions, which may not accurately reflect real-world conditions due to the lack of in-situ experimental datasets. To address these challenges and gaps, this paper presents a flexible hardware-in-the-loop (HIL) testbed for generating cyber-attack and physical fault datasets and demonstrating threat detection algorithms in a real building automation system (BAS) environment. This testbed combines hardware (i.e., real BAS with local HVAC controllers and a physical network) with software (i.e., high-fidelity models to represent behaviors of building envelope and HVAC energy systems), enabling emulations of realistic threats. Five HIL experiments, including one baseline without any threats, two with physical faults, and two with cyber-attacks, were conducted to generate datasets containing detailed network traffic and system states. A joint classification framework, incorporating a network analyzer and a physical HVAC fault detector, was proposed to automatically detect cyber-physical abnormalities on BAS at both the network and the physical HVAC levels. The network analyzer comprises a conditional random fields (CRF) based command validator and a statistics-based detection strategy. The fault detector employs a weather and schedule-based pattern matching and feature-based principal component analysis (WPM-FPCA) method. Evaluation of the classification using four metrics from the multi-class confusion matrix revealed an average accuracy of 90.2%, recall of 89.7%, precision of 88.5% and F1-score of 89.2%. Finally, these results demonstrate that the proposed joint classification framework can effectively differentiate between specific types of cyber-attacks (e.g., device reinitialization attack, network Denial-of-Service attack) and physical faults (e.g., air handling unit operational fault, cooling coil valve stuck) in real time for improved building energy management.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2023

Assessing thermal resilience of an assisted living facility during heat waves and cold snaps with power outages

Extreme hot and cold weather events are becoming more frequent, intense, and longer due to climate change. When these events occur coincidentally with power outages, the resulting extreme indoor temperatures pose a severe health hazard for occupants. This study conducted a holistic modeling and analysis of an assisted living facility, where senior residents live, to assess its thermal resilience performance under a six-day heat wave in 2015 and a three-day cold snap in 2021 with power outages. Impacts of 13 energy efficiency measures on thermal resilience and backup power capacity of the facility were evaluated. Three thermal resilience metrics: the SET (standard effective temperature) degree-hours, the Heat Index, and the Hours of Safety, were used and calculated from the EnergyPlus simulation models. Furthermore, major findings are: (1) the facility would suffer from extreme temperatures during the cold and hot events without a power supply, not meeting the passive survivability requirements; (2) most passive envelope measures improve thermal resilience for both hot and cold events, but making the building envelope airtight results in conflicting performance between the hot and cold events; (3) natural ventilation is an effective measure to mitigate summer indoor overheating; and (4) the energy efficiency package can reduce backup power capacity by 19% for the three-day cold snap. It is recommended that building technologies and design strategies be evaluated to consider co-benefits of energy use, thermal resilience, and backup power needs through building energy codes or policies for existing and new buildings, which are transitioning for decarbonization and climate resilience.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗
DOE OSTI2022

Domain architecture and catalysis of the Staphylococcus aureus fatty acid kinase

Fatty acid kinase (Fak) is a two-component enzyme that generates acyl-phosphate for phospholipid synthesis. Fak consists of a kinase domain protein (FakA) that phosphorylates a fatty acid enveloped by a fatty acid binding protein (FakB). The structural basis for FakB function has been established, but little is known about FakA. Here, we used limited proteolysis to define three separate FakA domains: the amino terminal FakA_N, the central FakA_L, and the carboxy terminal FakA_C. The isolated domains lack kinase activity, but activity is restored when FakA_N and FakA_L are present individually or connected as FakA_NL. The X-ray structure of the monomeric FakA_N captures the product complex with ADP and two Mg 2+ ions bound at the nucleotide site. The FakA_L domain encodes the dimerization interface along with conserved catalytic residues Cys240, His282, and His284. AlphaFold analysis of FakA_L predicts the catalytic residues are spatially clustered and pointing away from the dimerization surface. Furthermore, the X-ray structure of FakA_C shows that it consists of two subdomains that are structurally related to FakB. Analytical ultracentrifugation demonstrates that FakA_C binds FakB, and site-directed mutagenesis confirms that a positively charged wedge on FakB meshes with a negatively charged groove on FakA_C. Finally, small angle X-ray scattering analysis is consistent with freely rotating FakA_N and FakA_C domains tethered by flexible linkers to FakA_L. These data reveal specific roles for the three independently folded FakA protein domains in substrate binding and catalysis.

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

Engineering well-expressed, V2-immunofocusing HIV-1 envelope glycoprotein membrane trimers for use in heterologous prime-boost vaccine regimens

HIV-1 vaccine immunofocusing strategies may be able to induce broadly-reactive neutralizing antibodies (NAbs). Here, we engineered a panel of diverse, membrane-resident native HIV-1 trimers vulnerable to two broad targets—the V2 apex and fusion peptide (FP). Selection criteria included i) high expression and ii) infectious function, so that trimer neutralization sensitivity can be profiled in pseudovirus (PV) assays. Initially, we boosted the expression of 17 candidate trimers by truncating gp41 and introducing a gp120-gp41 SOS disulfide to prevent gp120 shedding. "Repairs" were made to fill glycan holes and eliminate other strain-specific aberrations. A new neutralization assay allowed PV infection when our standard assay was insufficient. Trimers with exposed V3 loops, a target of non-NAbs, were discarded. To try to increase V2-sensitivity, we removed clashing glycans and modified the C-strand. Notably, a D167N mutation improved V2-sensitivity in several cases. Glycopeptide analysis of JR-FL trimers revealed near complete sequon occupation and that filling the N197 glycan hole was well-tolerated. In contrast, sequon optimization and inserting/removing glycans at other positions frequently had global "ripple" effects on glycan maturation and sequon occupation throughout the gp120 outer domain and gp41. V2 MAb CH01 selectively bound to trimers with small high mannose glycans near the base of the V1 loop, thereby avoiding clashes. Knocking in a rare N49 glycan was found to perturb gp41 glycans, increasing FP NAb sensitivity—and sometimes improving expression. Finally, a biophysical analysis of VLPs revealed that i) ~25% of particles bear Env spikes, ii) spontaneous particle budding is high and only increases 4-fold upon Gag transfection, and iii) Env+ particles express ~30–40 spikes. Taken together, we identified 7 diverse trimers with a range of sensitivities to two targets to allow rigorous testing of immunofocusing vaccine concepts.

60 APPLIED LIFE SCIENCES↗