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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 379 records · Page 21
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

CRISPR–Cas9-mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

DNA nanostructures are a promising tool to deliver molecular payloads to cells. DNA origami structures, where long single-stranded DNA is folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here, we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR–Cas9 ribonucleoprotein binding sites on DNA nanostructures to increase shuttling into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA templates in human primary cells. Furthermore, our study validates virus-like particles as an efficient method of DNA nanostructure delivery, opening the possibility of delivering nanostructures in vivo to specific cell types. Together, these results provide new approaches to gene delivery with DNA nanostructures and establish their use as HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as biosensing, into cell nuclei.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Metadynamics simulations reveal mechanisms of Na + and Ca 2+ transport in two open states of the channelrhodopsin chimera, C1C2

Cation conducting channelrhodopsins (ChRs) are a popular tool used in optogenetics to control the activity of excitable cells and tissues using light. ChRs with altered ion selectivity are in high demand for use in different cell types and for other specialized applications. However, a detailed mechanism of ion permeation in ChRs is not fully resolved. Here, we use complementary experimental and computational methods to uncover the mechanisms of cation transport and valence selectivity through the channelrhodopsin chimera, C1C2, in the high- and low-conducting open states. Electrophysiology measurements identified a single-residue substitution within the central gate, N297D, that increased Ca 2+ permeability vs. Na + by nearly two-fold at peak current, but less so at stationary current. We then developed molecular models of dimeric wild-type C1C2 and N297D mutant channels in both open states and calculated the PMF profiles for Na + and Ca 2+ permeation through each protein using well-tempered/multiple-walker metadynamics. Results of these studies agree well with experimental measurements and demonstrate that the pore entrance on the extracellular side differs from original predictions and is actually located in a gap between helices I and II. Cation transport occurs via a relay mechanism where cations are passed between flexible carboxylate sidechains lining the full length of the pore by sidechain swinging, like a monkey swinging on vines. In the mutant channel, residue D297 enhances Ca 2+ permeability by mediating the handoff between the central and cytosolic binding sites via direct coordination and sidechain swinging. We also found that altered cation binding affinities at both the extracellular entrance and central binding sites underly the distinct transport properties of the low-conducting open state. This work significantly advances our understanding of ion selectivity and permeation in cation channelrhodopsins and provides the insights needed for successful development of new ion-selective optogenetic tools.

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

Aqueous-soluble bipyridine cobalt($\tiny{II}$/$\tiny{III}$) complexes act as direct redox mediators in photosystem I-based biophotovoltaic devices

Sustainable energy production is critical for meeting growing worldwide energy demands. Due to its stability and reduction potential, photosystem I (PSI) is attractive as the photosensitizer in biophotovoltaic devices. Herein, we characterize aqueous and organic solvent soluble synthetic bipyridine-based cobalt complexes as redox mediators for PSI-based biophotovoltaics applications. Cobalt-based complexes are not destructive to protein and have appropriate midpoint potentials for electron donation to PSI. We report on PSI stability in organic solvents commonly used in biophotovoltaics. We also show the effects of a mixed organic solvent phase on PSI reduction kinetics, slowing reduction rates approximately 8–38 fold as compared to fully aqueous systems, with implications for dye regeneration rates in PSI-based biophotovoltaics. Further, we show evidence of direct electron transfer from cobalt complexes to PSI. Finally, we report on photocurrent generation from Co mediator-PSI biophotovoltaic devices. Taken together, we discuss the development of novel Co complexes and our ability to fine-tune their characteristics via functional groups and counteranion choice to drive interaction with a biological electron acceptor on multiple levels from redox midpoints, spectral overlap, and solvent requirements, among others. This work suggests that fine-tuning of redox active species for interaction with a biological partner is possible for the creation and improvement of low cost, carbon-neutral energy production in the future.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Structure and regulation of human epithelial cell transforming 2 protein

Epithelial cell transforming 2 (Ect2) protein activates Rho GTPases and controls cytokinesis and many other cellular processes. Dysregulation of Ect2 is associated with various cancers. Here, we report the crystal structure of human Ect2 and complementary mechanistic analyses. The data show the C-terminal PH domain of Ect2 folds back and blocks the canonical RhoA-binding site at the catalytic center of the DH domain, providing a mechanism of Ect2 autoinhibition. Ect2 is activated by binding of GTP-bound RhoA to the PH domain, which suggests an allosteric mechanism of Ect2 activation and a positive-feedback loop reinforcing RhoA signaling. Furthermore this bimodal RhoA binding of Ect2 is unusual and was confirmed with Förster resonance energy transfer (FRET) and hydrogen–deuterium exchange mass spectrometry (HDX-MS) analyses. Several recurrent cancer-associated mutations map to the catalytic and regulatory interfaces, and dysregulate Ect2 in vitro and in vivo. Together, our findings provide mechanistic insights into Ect2 regulation in normal cells and under disease conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Engineering and Application of a Thermostable MHETase for PET Depolymerization

Enzymatic hydrolysis of poly(ethylene terephthalate) (PET) releases mono(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T m,app ) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043–5 was identified with a comparable thermostability (T m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077 LFK , which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

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

Structural and biochemical analyses of the nuclear IκBζ protein in complex with the NF-κB p50 homodimer

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.

Cell Biology↗
DOE OSTI2021

A Novel Platform for Algal Biomass Production Using Cellulosic Mixotrophy (CeMix) (Final Technical Report)

This novel project, branded CeMIX for cellulosic mixotrophy, targets solutions for several technological barriers limiting the deployment of algae cultivation in sunny, arid regions (specifically the southwest United States). Opening up the region for algae cultivation could increase DOE’s resource assessments and biofuel potential for the U.S. Mixotrophic metabolism in support of liquid fuel production is completely dependent on the availability of waste organic carbon in order to avoid diversion of food resources for fuel production. The use of cellulosic sugar hydrolysate (CSH) produced via the NREL process provided a standardized carbon source for study of the underlying biochemistry and metabolic adjustments to mixotrophy. Mixotrophic metabolism uses both photosynthetic CO 2 fixation and sugar oxidation to improve biomass productivity and harvest yield. The project was high-risk, high-reward in the sense that adding organic carbon to algal cultivation is an invitation for rapid-growth, heterotrophic contaminants to overtake the culture and consume mineral nutrients needed for algal growth. To help evade this outcome the project focused on acidophilic red algae that require low pH conditions with 40-48°C optimal temperature profiles that define them as low-range thermophiles. This is the only project in the BETO portfolio to utilize extreme conditions of low pH and high operating temperatures to control pathogens and competitors in mass culture. The project yielded five major outcomes. 1) Harvest densities of 5-10 g ash-free dry weight are easily achieved, providing a 10-fold reduction in water required and dewatering demand. 2) Catabolic repression of photosynthesis in G. sulphuraria is conditionally repressed by low oxygen. 3) Mixotrophic growth on cellulosic hydrolysate consumes all C6 and C5 sugars concomitant with increases in floridean starch with little change in lipid content. Protein content can be manipulated between 35 and 54 wt% by varying the C:N molar ratio from 10:1 to 20:1, respectively. 4) Mixotrophic cultures with up to 50 mM total sugar are remarkably stable at pH 2.5 and daily average reactor temperatures of 40°C. 5) Capital costs for glass tubular photobioreactors are prohibitive for liquid fuel production using a hydrothermal liquefaction processing pathway. The economic outlook for covered raceway ponds is better with a project minimum fuel selling price at $3.32 dm 3 GE -1 .

09 BIOMASS FUELS↗
DOE OSTI2024

Exploration of Tertiary Structure in Sequence-Defined Polymers Using Molecular Dynamics Simulations

Peptoids are a class of sequence-defined biomimetic polymers with peptide-like backbones and side chains located on backbone nitrogens rather than alpha carbons. These materials demonstrate a strong ability for precise control of single-chain structure, multiunit self-assembly, and macromolecular assembly through careful tuning of sequence due to the diversity of available side chains, although the driving forces behind these assemblies are often not understood. Prior experimental work has shown that linked 15mer peptoids can mimic the protein helical hairpin structure by leveraging the chirality-inducing nature of bulky side chains and hydrophobicity, but there are still gaps in our understanding of the relationship between sequence, stability, and particular secondary or tertiary structure. Here, we present a molecular dynamics (MD) study on the folding behavior of these polymers into hairpins, discussing the differences in structure from sequences with various characteristics in water and acetonitrile, and then compare the handedness preference of common helical motifs between solvents.

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

Mycobacterium tuberculosis CitA activity is modulated by cysteine oxidation and pyruvate binding

As an adaptation for survival during infection, Mycobacterium tuberculosis becomes dormant, reducing its metabolism and growth. Two types of citrate synthases have been identified in Mycobacterium tuberculosis, GltA2 and CitA. Previous work shows that overexpression of CitA, the secondary citrate synthase, stimulates the growth of Mycobacterium tuberculosis under hypoxic conditions without showing accumulation of triacylglycerols and makes mycobacteria more sensitive to antibiotics, suggesting that CitA may play a role as a metabolic switch during infection and may be an interesting TB drug target. To assess the druggability and possible mechanisms of targeting CitA with small-molecule compounds, the CitA crystal structure was solved to 2.1 Å by X-ray crystallography. The solved structure shows that CitA lacks an NADH binding site that would afford allosteric regulation, which is atypical of most citrate synthases. However, a pyruvate molecule is observed within the analogous domain, suggesting pyruvate may instead be the allosteric regulator for CitA. Further, the R149 and R153 residues forming the charged portion of the pyruvate binding pocket were mutated to glutamate and methionine, respectively, to assess the effect of mutations on activity. Protein thermal shift assay shows thermal stabilization of CitA in the presence of pyruvate compared to the two CitA variants designed to decrease pyruvate affinity. Solved crystal structures of both variants show no significant structural changes. However, the catalytic efficiency of the R153M variant increases by 2.6-fold. Additionally, we show that covalent modification of C143 of CitA by Ebselen completely arrests enzyme activity. Similar inhibition is observed using two spirocyclic Michael acceptor containing compounds, which inhibit CitA with IC app 50 values of 6.6 and 10.9 μM. A crystal structure of CitA modified by Ebselen was solved, but significant structural changes were lacking. Considering that covalent modification of C143 inactivates CitA and the proximity of C143 to the pyruvate binding site, this suggests that structural and/or chemical changes in this sub-domain are responsible for regulating CitA enzymatic activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Structural insights into acetylated histone ligand recognition by the $\mathrm{BDP1}$ bromodomain of Plasmodium falciparum

Plasmodium falciparum requires a two-host system, moving between Anopheles mosquito and humans, to complete its life cycle. To overcome such dynamic growth conditions its histones undergo various post-translational modifications to regulate gene expression. The P. falciparum Bromodomain Protein 1 (PfBDP1) has been shown to interact with acetylated lysine modifications on histone H3 to regulate the expression of invasion-related genes. Here, we investigated the ability of the PfBDP1 bromodomain to interact with acetyllsyine modifications on additional core and variant histones. A crystal structure of the PfBDP1 bromodomain (PfBDP1-BRD) reveals it contains the conserved bromodomain fold, but our comparative analysis between the PfBDP1-BRD and human bromodomain families indicates it has a unique binding mechanism. Solution NMR spectroscopy and ITC binding assays carried out with acetylated histone ligands demonstrate that it preferentially recognizes tetra-acetylated histone H4, and we detected weaker interactions with multi-acetylated H2A.Z in addition to the previously reported interactions with acetylated histone H3. Our findings indicate PfBDP1 may play additional roles in the P. falciparum life cycle, and the distinctive features of its bromodomain binding pocket could be leveraged for the development of new therapeutic agents to help overcome the continuously evolving resistance of P. falciparum against currently available drugs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Reversal of Catalytic Material Substrate Selectivity through Partitioning of Polymers in Hierarchically Ordered Virus-like Particle Frameworks

Control over the selectivity of catalytic materials is a topic of growing interest. Virus-like particle (VLP) based materials such as protein macromolecular frameworks (PMFs) are promising for catalytic applications due to their ease of assembly, modular ability to encapsulate a variety of enzymes, and ease of separation from a reaction mixture. Here we demonstrate the reversal of the initially negative material charge through the titration of a positively charged polymer into the material, causing the reversal of guest molecule uptake and enzymatic activity of PMFs. The charge-inverse material partitions a charged enzyme substrate to concentrate the substrate near an enzyme incorporated within the material, generating up to 5.9-fold increases in enzyme activity toward the partitioned substrate over the excluded substrate. Here we also show that the polymer distributes heterogeneously through the material up to a point of saturation and the effects of guest macromolecules on the lattice parameters of PMFs.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Mapping structural and dynamic divergence across the MBOAT family

Membrane-bound O-acyltransferases (MBOATs) are membrane-embedded enzymes that catalyze acyl chain transfer to a diverse group of substrates, including lipids, small molecules, and proteins. MBOATs share a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify lipid binding sites surrounding reactant gates in the surrounding membrane. Complementary bioinformatic analyses reveal a conserved role for re-entrant loop-2 in MBOAT fold stabilization and a key hydrogen bond bridging DGAT1 dimerization. Finally, we predict differences in MBOAT solvation and water gating properties. These data are pertinent to the design of MBOAT-specific inhibitors that encompass dynamic information within cellular mimetic environments.

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

Membrane Composition Influences Expression Yield of Plant Cytochrome P450s in E. coli Lysate-Based Cell-Free Systems

Plant cytochrome P450 enzymes are central to natural product biosynthesis, but remain difficult to express in microbial hosts due to their transmembrane nature. Lysate-based, cell-free expression systems allow supplementation with artificial membranes to support the expression and translocation of transmembrane proteins. We developed a framework to systematically test liposomal membrane compositions to enhance the plant P450 expression yield. Adjustments to common phospholipid ratios or the addition of plant galactolipids had minimal impact on expression. In contrast, blended liposomes containing Egg PC, sterol-conjugated phospholipids, and PEGylated lipids produced concentration-dependent increases in expression. Expression of an Escherichia coli mechanosensitive channel and three plant P450s improved more than 2-fold, with some P450s showing up to 14-fold enhancement. Furthermore, these findings highlight membrane composition as a key determinant of the P450 expression yield in cell-free expression systems. While P450 activity was not measured, these findings provide a framework for future workflows toward achieving functional plant transmembrane enzymes for the bioproduction of natural products.

TXTL↗
DOE OSTI2022

One fold, many functions—M23 family of peptidoglycan hydrolases

Bacterial cell walls are the guards of cell integrity. They are composed of peptidoglycan that provides rigidity to sustain internal turgor and ensures isolation from the external environment. In addition, they harbor the enzymatic machinery to secure cell wall modulations needed throughout the bacterial lifespan. The main players in this process are peptidoglycan hydrolases, a large group of enzymes with diverse specificities and different mechanisms of action. They are commonly, but not exclusively, found in prokaryotes. Although in most cases, these enzymes share the same molecular function, namely peptidoglycan hydrolysis, they are leveraged to perform a variety of physiological roles. A well-investigated family of peptidoglycan hydrolases is M23 peptidases, which display a very conserved fold, but their spectrum of lytic action is broad and includes both Gram- positive and Gram- negative bacteria. In this review, we summarize the structural, biochemical, and functional studies concerning the M23 family of peptidases based on literature and complement this knowledge by performing large-scale analyses of available protein sequences. This review has led us to gain new insight into the role of surface charge in the activity of this group of enzymes. We present relevant conclusions drawn from the analysis of available structures and indicate the main structural features that play a crucial role in specificity determination and mechanisms of latency. Our work systematizes the knowledge of the M23 family enzymes in the context of their unique antimicrobial potential against drug-resistant pathogens and presents possibilities to modulate and engineer their features to develop perfect antibacterial weapons.

Razew, Alicja↗
DOE OSTI2024

Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea

Pea (legume plant defense) phytoalexin pterocarpans (–)-maackiain and (+)-pisatin have opposite C6a and C11a configurations, but how this occurs biosynthetically is unknown. Herein, pea dirigent-protein (DP) PsPTS2, which generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), was investigated as regards stereoselectivity towards four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers. Each DMDI stereoisomer configuration was determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF, this being 20-fold faster than with the trans-(3R,4S)-isomer. The 4R-configured substrate’s near ß-axial OH orientation significantly enhanced its leaving group abilities in creating intermediate A-ring mono-quinone methide (QM), whereas the 4S-isomer’s 4-OH group α-equatorial orientation was a poorer leaving group. Substrate docking simulations also indicated that the 4R-configured ß-axial OH was closest to the Asp catalytic center, whereas the 4S-isomer’s α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S, 4R)-DMDIs were substrates, even with the former having the same C3/C4 stereochemistry as (+)-pisatin. PsPTS2, assayed with cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and its C3/C4 stereoisomers, gave 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF), with the same substrate stereoselectivity. DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and the tree legume Bolusanthus speciosus, as DMIF is found in both. PsPTS1 preferentially utilized cis-(3R,4R)-DMDI to give (–)-maackiain, this being 2200-fold more efficient than with cis-(3R,4R)-DMI. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, again reflecting the better 4R configured OH leaving group. Pea PsPTS2 and PsPTS1 provisionally provide a facile DP-engendered means to enable differing C6a and C11a configurations in (+)-pisatin and (–)-maackiain. Both DP transformations are considered to occur via DP-engendered generation of an identical mono-QM bound intermediate, which with PsPTS2 either re-aromatizes to give DMDIF or with PsPTS1 undergoes intramolecular cyclization to afford (–)-maackiain. Substrate docking simulations using PsPTS2 (2.5 Å resolution) and PsPTS1 (1.5 Å resolution) plausibly indicate that cis-(3R,4R)-DMDI binds in the anti-configuration in the PsPTS2 active site to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain.

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

Identification of 2-Hydroxyacyl-CoA Synthases with High Acyloin Condensation Activity for Orthogonal One-Carbon Bioconversion

One-carbon (C1) compounds are emerging as cost-effective and potentially carbon-negative feedstocks for biomanufacturing, which require efficient, versatile metabolic platforms for the synthesis of value-added products. Synthetic formyl-CoA elongation (FORCE) pathways allow diverse product synthesis from C1 compounds via iterative C1 elongation, operating independently from the host metabolism with reduced engineering complexity and improved theoretical yields. However, a major bottleneck was identified as the suboptimal kinetics of the core C1–C1 condensation enzyme, 2-hydroxyacyl-CoA synthase (HACS), catalyzing the acyloin condensation reaction between formaldehyde and formyl-CoA. Furthermore, we used a combinatorial approach of bioprospecting and rational protein engineering to identify multiple HACS variants with significantly improved activities toward C1 substrates. Sequence and structure alignment of the active variants elucidated the key regions for the catalytic function, which were targeted for mutagenesis, leading to improved catalytic efficiency. In parallel, a consecutive round of bioprospecting for homologs with high similarity with active variants revealed a highly active HACS variant exhibiting up to 7-fold improvement in catalytic efficiency (k cat /K M ) and 14-fold improvement in the FORCE pathway flux in vivo compared to the previous reports. Upon further optimization of the downstream pathway, the orthogonal C1-to-product bioconversion system showed a metabolic flux of up to 700 μM glycolate OD –1 h –1 (2.1 mmol gDCW –1 h –1 ) and an industrially relevant glycolate titer, rate, and yield of 5.2 g L –1 (67.8 mM), 0.22 g L –1 h –1 , and 94% carbon yield, respectively.

2-hydroxyacyl-CoA synthase↗
DOE OSTI2021

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate

Background: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. Methods: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. Results: Purified nHRP2 was identified by SDS-PAGE and western blot as a –60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. Conclusions: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Tau and Membranes: Interactions That Promote Folding and Condensation

Tau misfolding and assembly is linked to a number of neurodegenerative diseases collectively described as tauopathies, including Alzheimer’s disease (AD) and Parkinson’s disease. Anionic cellular membranes, such as the cytosolic leaflet of the plasma membrane, are sites that concentrate and neutralize tau, primarily due to electrostatic interactions with tau’s microtubule binding repeat domain (RD). In addition to electrostatic interactions with lipids, tau also has interactions with membrane proteins, which are important for tau’s cellular functions. Tau also interacts with lipid tails to facilitate direct translocation across the membrane and can form stable protein-lipid complexes involved in cell-to-cell transport. Concentrated tau monomers at the membrane surface can form reversible condensates, change secondary structures, and induce oligomers, which may eventually undergo irreversible crosslinking and fibril formation. These β-sheet rich tau structures are capable of disrupting membrane organization and are toxic in cell-based assays. Given the evidence for relevant membrane-based tau assembly, we review the emerging hypothesis that polyanionic membranes may serve as a site for phase-separated tau condensation. Membrane-mediated phase separation may have important implications for regulating tau folding/misfolding, and may be a powerful mechanism to spatially direct tau for native membrane-mediated functions.

59 BASIC BIOLOGICAL SCIENCES↗