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DOE OSTI2021

Cell specific photoswitchable agonist for reversible control of endogenous dopamine receptors

Dopamine controls diverse behaviors and their dysregulation contributes to many disorders. Our ability to understand and manipulate the function of dopamine is limited by the heterogenous nature of dopaminergic projections, the diversity of neurons that are regulated by dopamine, the varying distribution of the five dopamine receptors (DARs), and the complex dynamics of dopamine release. In order to improve our ability to specifically modulate distinct DARs, here we develop a photo-pharmacological strategy using a Membrane anchored Photoswitchable orthogonal remotely tethered agonist for the Dopamine receptor (MP-D). Our design selectively targets D1R/D5R receptor subtypes, most potently D1R (MP-D1 ago ), as shown in HEK293T cells. In vivo, we targeted dorsal striatal medium spiny neurons where the photo-activation of MP-D1 ago increased movement initiation, although further work is required to assess the effects of MP-D1 ago on neuronal function. Our method combines ligand and cell type-specificity with temporally precise and reversible activation of D1R to control specific aspects of movement. Our results provide a template for analyzing dopamine receptors.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

The regulation of the cell wall by glycosylphosphatidylinositol-anchored proteins in Arabidopsis

A polysaccharides-based cell wall covers the plant cell, shaping it and protecting it from the harsh environment. Cellulose microfibrils constitute the cell wall backbone and are embedded in a matrix of pectic and hemicellulosic polysaccharides and glycoproteins. Various environmental and developmental cues can regulate the plant cell wall, and diverse glycosylphosphatidylinositol (GPI)-anchored proteins participate in these regulations. GPI is a common lipid modification on eukaryotic proteins, which covalently tethers the proteins to the membrane lipid bilayer. Catalyzed by a series of enzymic complexes, protein precursors are post-translationally modified at their hydrophobic carboxyl-terminus in the endomembrane system and anchored to the lipid bilayer through an oligosaccharidic GPI modification. Ultimately, mature proteins reach the plasma membrane via the secretory pathway facing toward the apoplast and cell wall in plants. In Arabidopsis, more than three hundred GPI-anchored proteins (GPI-APs) have been predicted, and many are reported to be involved in diverse regulations of the cell wall. In this review, we summarize GPI-APs involved in cell wall regulation. GPI-APs are proposed to act as structural components of the cell wall, organize cellulose microfibrils at the cell surface, and during cell wall integrity signaling transduction. Besides regulating protein trafficking, the GPI modification is potentially governed by a GPI shedding system that cleaves and releases the GPI-anchored proteins from the plasma membrane into the cell wall.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Ion Specificity Influences on the Structure of Zwitterionic Brushes

Zwitterionic brushes have a wide range of applications, including use as lubricating surfaces and antifouling membranes. In this study, densely end-tethered poly(cysteine methacrylate) (PCysMA) brushes were synthesized using surfaceinitiated activators regenerated by electron transfer atom transfer radical polymerization (SI-ARGET-ATRP). The structure of the PCysMA brushes was investigated using ellipsometry, X-ray reflectivity (XRR), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and atomic force microscopy (AFM). The results of these characterization techniques were used to study the effect of SO 4 2– , Cl – , NO 3 – , Br – , and SCN – anions, divalent Ca 2+ and Ba 2+ cations, and trivalent Y 3+ cations on the structure of the PCysMA brushes. The results showed that the PCysMA brushes in solution exhibit an “antipolyelectrolyte” effect to a certain degree, which inversely follows the Hofmeister series of anions. The introduction of divalent cations Ca 2+ and Ba 2+ had a modest impact on the dimensions of the PCysMA brushes, indicating that chelating interactions between the cations and zwitterion units work against the “antipolyelectrolyte” effect. The complexation was even stronger in the presence of trivalent Y 3+ cations, which caused the PCysMA brushes to shrink. These findings highlight the importance of ion specificity to the structure of zwitterionic brushes in aqueous solutions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

A Review of the Use of Immobilized Ionic Liquids in the Electrochemical Conversion of CO2

This paper is a review on the application of imidazolium-based ionic liquids tethered to polymer backbones in the electrochemical conversion of CO2 to carbon monoxide and formic acid. These tethered ionic liquids have been incorporated into novel anion ion exchange membranes for CO2 electrolysis, as well as for ionomers that have been incorporated into the cathode catalyst layer, providing a co-catalyst for the reduction reaction. In using these tethered ionic liquids in the cathode catalyst composition, the cell operating current increased by a factor of two or more. The Faradaic efficiencies also increased by 20–30%. This paper provides a review of the literature, in addition to providing some new experimental results from Dioxide Materials, in the electrochemical conversion of CO2 to CO and formic acid.

09 BIOMASS FUELS↗

DOE OSTI2023

Optical Control of Dopamine D2-like Receptors with Cell-Specific Fast-Relaxing Photoswitches

Dopamine D2-like receptors (D2R, D3R, and D4R) control diverse physiological and behavioral functions and are important targets for the treatment of a variety of neuropsychiatric disorders. Their complex distribution and activation kinetics in the brain make it difficult to target specific receptor populations with sufficient precision. We describe a new toolkit of light-activatable, fast-relaxing, covalently taggable chemical photoswitches that fully activate, partially activate, or block D2-like receptors. This technology combines the spatiotemporal precision of a photoswitchable ligand (P) with cell type and spatial specificity of a genetically encoded membrane anchoring protein (M) to which the P tethers. These tools set the stage for targeting endogenous D2-like receptor signaling with molecular, cellular, and spatiotemporal precision using only one wavelength of light.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Mechanism of tethered agonist-mediated signaling by polycystin-1

Mutations of polycystin-1 (PC1) are the major cause (85% of cases) of autosomal dominant polycystic kidney disease (ADPKD), which is the fourth leading cause of kidney failure. PC1 is thought to function as an atypical G protein-coupled receptor, yet the mechanism by which PC1 regulates G-protein signaling remains poorly understood. A significant portion of ADPKD mutations of PC1 encode a protein with defects in maturation or reduced function that may be amenable to functional rescue. In this work, we have combined complementary biochemical and cellular assay experiments and accelerated molecular simulations, which revealed an allosteric transduction pathway in activation of the PC1 C-terminal fragment. Our findings will facilitate future rational drug design efforts targeting the PC1 signaling function. Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074–S3585 and R3848–E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell-membranes to model association reactions of proteins with lipid-bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal-Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers, deposited on the interior surfaces of wide-pore silica surfaces. We examine the question whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin-coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ~60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. Here, the results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.

36 MATERIALS SCIENCE↗

DOE OSTI2020

Assembly of Building Blocks by Double-End-Anchored Polymers in the Dilute Regime Mediated by Hydrophobic Interactions at Controlled Distances

Hierarchical assembly of building blocks via competing, orthogonal interactions is a hallmark of many of nature’s composite materials that do not require highly specific ligand-receptor interactions. To mimic this assembly mechanism requires the development of building blocks capable of tunable interactions. In the present work, we explored the interplay between repulsive (steric and electrostatic) and attractive hydrophobic forces. The designed building blocks allow hydrophobic forces to effectively act at controlled, large distances, to create and tune the assembly of membrane-based building blocks under dilute conditions and affect their interactions with cellular membranes via physical cross-bridges. Specifically, we employed double-end-anchored poly(ethylene glycol)s (DEA-PEGs)—hydrophilic PEG tethers with hydrophobic tails on both ends. Using differential-interference-contrast optical microscopy, synchrotron small angle X-ray scattering (SAXS), and cryogenic electron microscopy, we investigated the ability of DEA-PEGs to mediate assembly in the dilute regime on multiple length scales and on practical time scales. The PEG length, anchor hydrophobicity, and molar fraction of DEA-PEG molecules within a membrane strongly affect the assembly properties. Additional tuning of the intermembrane interactions can be achieved by adding repulsive interactions via PEG-lipids (steric) or cationic lipids to the DEA-PEG-mediated attractions. Further, while the optical and electron microscope imaging methods provided qualitative evidence of the ability of DEA-PEGs to assemble liposomes, the SAXS measurements and quantitative line-shape analysis in dilute preparations demonstrated that the ensemble average of loosely organized liposomal assemblies maintains DEA-PEG concentration-dependent tethering on defined nanometer length scales. For cationic liposome–DNA nanoparticles (CL–DNA NPs), aggregation induced by DEA-PEGs decreased internalization of NPs by cells, but tuning the DEA-PEG-induced attractions by adding repulsive steric interactions via PEG-lipids limited aggregation and increased NP uptake. However, confocal microscopy imaging together with colocalization studies with Rab11 and LysoTracker as markers of intracellular pathways showed that modifying CL–DNA NPs with DEA-PEGs alters their interactions with the plasma and endosomal membranes.

- x-ray scattering↗

DOE OSTI2021

Characterization of suspended membrane waveguides towards a photonic atom trap integrated platform

We demonstrate an optical waveguide device, capable of supporting the high, in-vacuum, optical power necessary for trapping a single atom or a cold atom ensemble with evanescent fields. Our photonic integrated platform, with suspended membrane waveguides, successfully manages optical powers of 6 mW (500 μ m span) to nearly 30 mW (125 μ m span) over an un-tethered waveguide span. This platform is compatible with laser cooling and magneto-optical traps (MOTs) in the vicinity of the suspended waveguide, called the membrane MOT and the needle MOT, a key ingredient for efficient trap loading. We evaluate two novel designs that explore critical thermal management features that enable this large power handling. This work represents a significant step toward an integrated platform for coupling neutral atom quantum systems to photonic and electronic integrated circuits on silicon.

36 MATERIALS SCIENCE↗

DOE OSTI2021

Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5

Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Leading the way out: how a plant myosin facilitates vesicle tethering during exocytosis

Exocytosis is a process by which molecules, such as lipids, proteins, and polysaccharides, are delivered to the plasma membrane (PM) and the extracellular space via vesicular transport. Precise orchestration of this process ensures the spatiotemporal specificity of cargo delivery and therefore plays a vital role in cell growth, function, and response to environmental cues (reviewed in Wu and Guo, 2015; Ravikumar et al., 2017). Lastly, while sophisticated mechanisms for exocytosis have been described in animal and yeast cells, the extent of conservation of these mechanisms in plant systems is far less clear.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Blocking C-terminal processing of KRAS4b via a direct covalent attack on the CaaX-box cysteine

RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hypervariable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top–down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-α3-α4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.

C185↗

DOE OSTI2022

Thermally stable hydrocarbon-based anion exchange membrane and ionomers

An anion exchange membrane is composed of a copolymer of 1,1-diphenylethylene and one or more styrene monomers, such as 4-tert-butylstyrene. The copolymer includes a backbone substituted with a plurality of ionic groups coupled to phenyl groups on the backbone via hydrocarbyl tethers between about 1 and about 7 carbons in length. High-temperature conditions enabled by these copolymers enhance conductivity performance, making them particularly suitable for use in anion exchange membranes in fuel cells, electrolyzers employing hydrogen, ion separations, etc. The properties of the membranes can be tuned via the degree of functionalization of the phenyl groups and selection of the functional groups, such as quaternary ammonium groups. Several processes can be used to incorporate the desired ionic functional groups into the polymers, such as chloromethylation, radical bromination, Friedel-Crafts acylation and alkylation, sulfonation followed by amination, or combinations thereof.

Lee, Sangwoo↗

DOE OSTI2024

Thermally stable hydrocarbon-based anion exchange membrane and ionomers

Lee, Sangwoo↗

DOE OSTI2024

Activation of polycystin-1 signaling by binding of stalk-derived peptide agonists

Polycystin-1 (PC1) is the protein product of thePKD1gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.

Life Sciences & Biomedicine - Other Topics↗

DOE OSTI2022

Imidazolium-Type Anion Exchange Membranes for Improved Organic Acid Transport and Permselectivity in Electrodialysis

Most commercial anion exchange membranes (AEMs) deploy quaternary ammonium moieties. Alternative cation moieties have been explored in AEMs for fuel cells, but there are no studies focused examining alternative tethered cations in AEMs for ionic separations—such as organic acid anion transport via electrodialysis. H-cell and conductivity experiments demonstrate that tethered benzyl 1-methyl imidazolium groups in polysulfone AEMs enhance lactate conductivity by 49% and improved lactate anion flux by 24x when compared to a quaternary benzyl ammonium polysulfone AEM. An electrodialysis demonstration with the imidazolium-type AEM showed a 2x improvement in lactate anion flux and 20% improvement in permselectivity when benchmarked against the quaternary ammonium AEM. Molecular dynamics and 2D NOESY NMR revealed closer binding of lactate anions to the imidazolium cations when compared to the quaternary ammonium cation. It is posited that this closer binding is responsible to greater flux values observed with imidazolium-type AEM.

36 MATERIALS SCIENCE↗

DOE OSTI2024

Understanding and Manipulating Counterion Condensation within Charged Polymer Electrolytes for Selective and Low Resistant Membrane Separations

Polymeric ion-exchange membranes are found at the heart of numerous electrochemical systems spanning separations (electrodialysis and membrane capacitive deionization) and energy conversion and storage (flow batteries, fuel cells, and electrolyzers). The ion-exchange membrane separators need to be ion conducting but electron insulating, and in many scenarios selective for a particular ion to conduct. Because of their polymeric nature, the molecular architectures of ion-exchange membranes lack the molecular precision found in many inorganic materials. Plus, they contain numerous structural defects that impede ionic transport. As a result of their poor structure, it has been difficult to carefully probe the underlying physics that governs ion transport in ion-exchange membranes. We investigated counterion condensation phenomena in ion-exchange membranes and correlated it to bulk ionic charge transport and selectivity in precisely defined and long-range ordered materials afforded through the principles of directed self-assembly. A multi-faceted approach spanning i.) advanced metrology, two-dimensional atomic force microscopy, x-ray scattering, and ion-exchange equilibria, and ii.) molecular simulations (classical molecular dynamics and ab-inito molecular dynamics) that bridge multiple time and length scales, were employed to correlate the extent of counterion condensation to ionic conductivity and selectivity. After uncovering the extent of the condensation phenomena in this project, new macromolecular chemistries were employed to manipulate the ion pairing interactions of the condensed counterions to tethered ions on the polymer backbone so more facile and selective conduction rates can be attained. The research has aided in the rational design of polymeric ion-exchange membrane separators with high current density for electrochemical systems and subsequently better energy efficiency.

36 MATERIALS SCIENCE↗