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At least 397 records · Page 22
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Pyoverdine binding aptamers and label-free electrochemical detection of pseudomonads

Pyoverdines are iron-chelating siderophores employed by various pseudomonads to promote their growth in iron-limited environments, facilitating both beneficial and detrimental interactions with co-inhabiting microbes or hosts, including plants and animals. The fluorescent pseudomonads produce fluorescent pyoverdines comprised of a conserved central chromophore and a unique strain-specific peptidic side chain produced by non-ribosomal peptide synthetases. Pyoverdine Pf5 (PVD-Pf5) is produced by Pseudomonas protegens Pf-5, a species known for supporting plant growth and its involvement in plant pathogen control. To develop a means of exploring the dynamics of P. protegens activity in soil and in the rhizosphere, we selected DNA aptamers that specifically recognize PVD-Pf5 with high affinities. Two selected aptamers with only 16% identity in sequence were examined for structure and function. We found evidence that both aptamers form structures in their apo-forms and one aptamer has structural features suggesting the presence of a G-quadruplex. Although their tertiary structures are predicted to be different, both aptamers bind the target PVD-Pf5 with similar affinities and do not bind other siderophores, including the related pyoverdine, pseudobactin, produced by Pseudomonas sp. B10. One aptamer binds the pyoverdine peptide component and may also interact with the chromophore. This aptamer was integrated into a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PVD-Pf5 and not to detect other siderophores that do not bind to the aptamer when evaluated in solution. This sensor provides a future opportunity to track the locations of P. protegens around plant roots and to monitor PVD-Pf5 production and movement through the soil.

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

Solvent Acts as the Referee in a Match‐Up Between Charged and Preorganized Receptors

The prevalence of anion-cation contacts in biomolecular recognition under aqueous conditions suggests that ionic interactions should dominate the binding of anions in solvents across both high and low polarities. Investigations of this idea using titrations in low polarity solvents are impaired by interferences from ion pairing that prevent a clear picture of binding. To address this limitation and test the impact of ion-ion interactions across multiple solvents, we quantified chloride binding to a cationic receptor after accounting for ion pairing. In these studies, we created a chelate receptor using aryl-triazole CH donors and a quinolinium unit that directs its cationic methyl inside the binding pocket. In low-polarity dichloromethane, the 1 : 1 complex (log K 1 : 1 ~ 7.3) is more stable than neutral chelates, but fortuitously comparable to a preorganized macrocycle (log K 1 : 1 ~ 6.9). Polar acetonitrile and DMSO diminish stabilities of the charged receptor (log K 1 : 1 ~ 3.7 and 1.9) but surprisingly 100-fold more than the macrocycle. While both receptors lose stability by dielectric screening of electrostatic stability, the cationic receptor also pays additional costs of organization. Thus even though the charged receptor has stronger binding in apolar solvents, the uncharged receptor has more anion affinity in polar solvents.

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

Symmetry breaking of fluorophore binding to a G-quadruplex generates an RNA aptamer with picomolar K D

Fluorogenic RNA aptamer tags with high affinity enable RNA purification and imaging. The G-quadruplex (G4) based Mango (M) series of aptamers were selected to bind a thiazole orange based (TO1-Biotin) ligand. Using a chemical biology and reselection approach, we have produced a MII.2 aptamer–ligand complex with a remarkable set of properties: Its unprecedented K D of 45 pM, formaldehyde resistance (8% v/v), temperature stability and ligand photo-recycling properties are all unusual to find simultaneously within a small RNA tag. Crystal structures demonstrate how MII.2, which differs from MII by a single A23U mutation, and modification of the TO1-Biotin ligand to TO1-6A-Biotin achieves these results. MII binds TO1-Biotin heterogeneously via a G4 surface that is surrounded by a stadium of five adenosines. Breaking this pseudo-rotational symmetry results in a highly cooperative and homogeneous ligand binding pocket: A22 of the G4 stadium stacks on the G4 binding surface while the TO1-6A-Biotin ligand completely fills the remaining three quadrants of the G4 ligand binding face. Similar optimization attempts with MIII.1, which already binds TO1-Biotin in a homogeneous manner, did not produce such marked improvements. We use the novel features of the MII.2 complex to demonstrate a powerful optically-based RNA purification system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

A molecular mechanism for the generation of ligand-dependent differential outputs by the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Enhanced sensitivity for electron affinity measurements of rare elements

The electron affinity (EA), the energy released when a neutral atom binds an additional electron, is a fundamental property of atoms that is governed by electron-electron correlations and is strongly related to an element’s chemical reactivity. However, conventional techniques for EA determination lack the experimental sensitivity to probe very scarce samples. As a result, the EA for the heaviest elements of the periodic table is entirely uncharted. Here, we present a novel technique to determine EAs through Laser Photodetachment Threshold Spectroscopy, performed in an electrostatic ion beam trap to increase the samples’ exposure to laser photons and, thus, improve the experimental signal sensitivity by three orders of magnitude. Moreover, the additional exposure time allows the use of lower-power continuous-wave narrow-band lasers that reduce uncertainties associated with broadening effects induced by the laser bandwidth. By applying this technique, we measure the EA of 35 Cl to be 3.612720(44) eV, achieving state-of-the-art precision while employing five orders of magnitude fewer anions. The demonstrated sensitivity paves the way for systematic EA measurements across isotopic chains - including isotope shifts and hyperfine splittings - and ultimately for the first direct determination of electron affinities in superheavy elements.

Maier, F. M. [Univ. of Greifswald (Germany); Europ↗
DOE OSTI2024

Vaccination induces broadly neutralizing antibody precursors to HIV gp41

A key barrier to the development of vaccines that induce broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV) and other viruses of high antigenic diversity is the design of priming immunogens that induce rare bnAb-precursor B cells. The high neutralization breadth of the HIV bnAb 10E8 makes elicitation of 10E8-class bnAbs desirable; however, the recessed epitope within gp41 makes envelope trimers poor priming immunogens and requires that 10E8-class bnAbs possess a long heavy chain complementarity determining region 3 (HCDR3) with a specific binding motif. We developed germline-targeting epitope scaffolds with affinity for 10E8-class precursors and engineered nanoparticles for multivalent display. Scaffolds exhibited epitope structural mimicry and bound bnAb-precursor human naive B cells in ex vivo screens, protein nanoparticles induced bnAb-precursor responses in stringent mouse models and rhesus macaques, and mRNA-encoded nanoparticles triggered similar responses in mice. Thus, germline-targeting epitope scaffold nanoparticles can elicit rare bnAb-precursor B cells with predefined binding specificities and HCDR3 features.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40 Ub -Lys92 Ube2N ) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme’s true physiological substrate.

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

Multivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC 50 ) values as low as 9 × 10 - 14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Conformation-specific synthetic intrabodies modulate mTOR signaling with subcellular spatial resolution

Subcellular compartmentalization is integral to the spatial regulation of mechanistic target of rapamycin (mTOR) signaling. However, the biological outputs associated with location-specific mTOR signaling events are poorly understood and challenging to decouple. Here, we engineered synthetic intracellular antibodies (intrabodies) that are capable of modulating mTOR signaling with genetically programmable spatial resolution. Epitope-directed phage display was exploited to generate high affinity synthetic antibody fragments (Fabs) against the FKBP12–Rapamycin binding site of mTOR (mTOR FRB ). We determined high-resolution crystal structures of two unique Fabs that discriminate distinct conformational states of mTOR FRB through recognition of its substrate recruitment interface. By leveraging these conformation-specific binders as intracellular probes, we uncovered the structural basis for an allosteric mechanism governing mTOR complex 1 (mTORC1) stability mediated by subtle structural adjustments within mTOR FRB . Furthermore, our results demonstrated that synthetic binders emulate natural substrates by employing divergent yet complementary hydrophobic residues at defined positions, underscoring the broad molecular recognition capability of mTOR FRB . Intracellular signaling studies showed differential time-dependent inhibition of S6 kinase 1 and Akt phosphorylation by genetically encoded intrabodies, thus supporting a mechanism of inhibition analogous to the natural product rapamycin. Finally, we implemented a feasible approach to selectively modulate mTOR signaling in the nucleus through spatially programmed intrabody expression. These findings establish intrabodies as versatile tools for dissecting the conformational regulation of mTORC1 and should be useful to explore how location-specific mTOR signaling influences disease progression.

Science & Technology - Other Topics↗
DOE OSTI2022

Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant of concern evades antibody-mediated immunity that comes from vaccination or infection with earlier variants due to accumulation of numerous spike mutations. To understand the Omicron antigenic shift, we determined cryo–electron microscopy and x-ray crystal structures of the spike protein and the receptor-binding domain bound to the broadly neutralizing sarbecovirus monoclonal antibody (mAb) S309 (the parent mAb of sotrovimab) and to the human ACE2 receptor. We provide a blueprint for understanding the marked reduction of binding of other therapeutic mAbs that leads to dampened neutralizing activity. Remodeling of interactions between the Omicron receptor-binding domain and human ACE2 likely explains the enhanced affinity for the host receptor relative to the ancestral virus.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Chelating platform for delivery of radionuclides

Siderocalin-metal chelator combinations that bind metallic radioisotopes used in nuclear medicine with high affinity are described. The high affinity siderocalin-metal chelator combinations include a number of chelator backbone arrangements with functional groups that coordinate with metals. The siderocalin-metal chelator combinations can be used to deliver radionuclides for imaging and therapeutic purposes.

Strong, Roland K.↗
DOE OSTI2023

Synthetic anti-plague antibodies

Monoclonal antibodies that specifically bind the F1 antigen of Yersinia pestis with high affinity are described. Use of the monoclonal antibodies for the detection of Y. pestis infection and the diagnosis of plague are also described. Immunoconjugates of the monoclonal antibodies and a radionuclide can also be used for the treatment of a Y. pestis infection.

Lillo, Antonietta Maria↗
DOE OSTI2024

A protein phosphatase 1 specific phos phatase ta rgeting p eptide (PhosTAP) to identify the PP1 phosphatome

Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.

Science & Technology - Other Topics↗
DOE OSTI2022

Rational design of photosynthetic reaction center protein maquettes

New technologies for efficient solar-to-fuel energy conversion will help facilitate a global shift from dependence on fossil fuels to renewable energy. Nature uses photosynthetic reaction centers to convert photon energy into a cascade of electron-transfer reactions that eventually produce chemical fuel. The design of new reaction centers de novo deepens our understanding of photosynthetic charge separation and may one day allow production of biofuels with higher thermodynamic efficiency than natural photosystems. Recently, we described the multi-step electron-transfer activity of a designed reaction center maquette protein (the RC maquette), which can assemble metal ions, tyrosine, a Zn tetrapyrrole, and heme into an electron-transport chain. Here, we detail our modular strategy for rational protein design and show that the intended RC maquette design agrees with crystal structures in various states of assembly. A flexible, dynamic apo-state collapses by design into a more ordered holo-state upon cofactor binding. Crystal structures illustrate the structural transitions upon binding of different cofactors. Spectroscopic assays demonstrate that the RC maquette binds various electron donors, pigments, and electron acceptors with high affinity. We close with a critique of the present RC maquette design and use electron-tunneling theory to envision a path toward a designed RC with a substantially higher thermodynamic efficiency than natural photosystems.

09 BIOMASS FUELS↗
DOE OSTI2024

Structural mechanisms for binding and activation of a contact-quenched fluorophore by RhoBAST

The fluorescent light-up aptamer RhoBAST, which binds and activates the fluorophore–quencher conjugate tetramethylrhodamine-dinitroaniline with high affinity, super high brightness, remarkable photostability, and fast exchange kinetics, exhibits excellent performance in super-resolution RNA imaging. Here we determine the co-crystal structure of RhoBAST in complex with tetramethylrhodamine-dinitroaniline to elucidate the molecular basis for ligand binding and fluorescence activation. The structure exhibits an asymmetric “A”-like architecture for RhoBAST with a semi-open binding pocket harboring the xanthene of tetramethylrhodamine at the tip, while the dinitroaniline quencher stacks over the phenyl of tetramethylrhodamine instead of being fully released. Molecular dynamics simulations show highly heterogeneous conformational ensembles with the contact-but-unstacked fluorophore–quencher conformation for both free and bound tetramethylrhodamine-dinitroaniline being predominant. The simulations also show that, upon RNA binding, the fraction of xanthene-dinitroaniline stacked conformation significantly decreases in free tetramethylrhodamine-dinitroaniline. This highlights the importance of releasing dinitroaniline from xanthene tetramethylrhodamine to unquench the RhoBAST–tetramethylrhodamine-dinitroaniline complex. Using SAXS and ITC, we characterized the magnesium dependency of the folding and binding mode of RhoBAST in solution and indicated its strong structural robustness. The structures and binding modes of relevant fluorescent light-up aptamers are compared, providing mechanistic insights for rational design and optimization of this important fluorescent light-up aptamer-ligand system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

ModE Regulates Alternative Nitrogenase Expression in the Methanogen Methanosarcina acetivorans

ABSTRACT All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans , also contain alternative vanadium‐ and iron‐nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo‐nitrogenase activity. The factors that control the expression of the alternative nitrogenases in response to Mo availability are unknown in methanogens. Here we show that ModE is the regulator that represses transcription of the vnf and anf operons in M. acetivorans when cells are grown with Mo. CRISPRi repression of modE results in a significant increase in the transcription of the vnf and anf operons as well as the detection of Fe‐nitrogenase during nitrogen fixation in the presence of Mo. Gel shift assays with recombinant ModE demonstrated that ModE binds a specific sequence motif upstream of the vnf and anf operons, as well as other genes and operons related to nitrogen fixation and Mo transport. However, purified ModE does not contain Mo, and the addition of Mo does not alter the affinity of ModE for DNA, indicating M. acetivorans ModE may not directly bind Mo. This study shows that ModE is the primary Mo‐responsive regulator of alternative nitrogenase expression in M. acetivorans , but other factor(s) are likely involved in directly sensing Mo.

Chanderban, Melissa [Department of Biological Scie↗
DOE OSTI2022

Development of high-affinity nanobodies specific for Na V 1.4 and Na V 1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na V s, are responsible for the rapid rise of action potentials in excitable tissues. Na V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the Na V 1.4 (skeletal muscle) and Na V 1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na V 1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na V isoform has been challenging because they usually show undesired crossreactivity for different Na V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa V 1.4 or CTNa V 1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa V 1.4 and CTNa V 1.5 with high affinity (K D ~ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na V 1.4 and Na V 1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na V 1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na V reagents to capture Na V s from cell lysates and as molecular visualization agents for Na V s.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Structures of drug-specific monoclonal antibodies bound to opioids and nicotine reveal a common mode of binding

Opioid-related fatal overdoses have reached epidemic proportions. Because existing treatments for opioid use disorders offer limited long-term protection, accelerating the development of newer approaches is critical. Monoclonal antibodies (mAbs) are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier, thus preventing or reversing opioid toxicity. We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine. To determine their binding mechanism, we used X-ray crystallography to solve the structures of mAbs bound to their respective targets, to 2.2 Å resolution or higher. Structural analysis showed a critical convergent hydrogen bonding mode that is dependent on a glutamic acid residue in the mAbs’ heavy chain and a tertiary amine of the ligand. Further, characterizing drug-mAb complexes represents a significant step toward rational antibody engineering and future manufacturing activities to support clinical evaluation.

60 APPLIED LIFE SCIENCES↗