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At least 631 records · Page 35
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na + sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (K D ) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Solution characterization of the dynamic conjugative entry exclusion protein TraG

The R100 plasmid and the secretion system it encodes are representative of F-like conjugative type IV secretion systems for the transmission of mobile DNA elements in gram-negative bacteria, serving as a major contributor to the spread of antibiotic resistance in bacterial pathogens. The TraG protein of F-like systems consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through a process termed entry exclusion. In the donor cell, it interacts with TraN to facilitate mating pair stabilization; however, if a mating pore forms between bacteria with identical plasmids, TraG* interacts with its cognate TraS in the inner membrane of the recipient bacterium to prevent redundant donor–donor conjugation. Structural studies of TraG* from the R100 plasmid have revealed the presence of a dynamic region between the N- and C-terminal domains of TraG. Thermofluor, circular dichroism, collision-induced unfolding–mass spectrometry, and size exclusion chromatography linked to multiangle light scattering and small angle x-ray scattering experiments indicated an N-terminal truncation mutant displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* are hypothesized to serve as part of a flexible linker between the two independently functioning domains.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2004

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LNI) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered Trademark) Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark) software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Hammond, Dianne K.↗
NASA NTRS2004

Antigenic Protein In Microgravity-Grown Human Mixed Mullerian Tumor (LN1) Cells Preserved In RNA Stabilizing Agent

Cells treated with RNAlater(TradeMark) have previously been shown to contain antigenic proteins that can be visualized using Western blot analysis. These proteins seem to be stable for several months when stored in RNA stabilizer at 4 C. Antigenic protein can be recovered from cells that have been processed using an Ambion RNAqueous(Registered TradeMark) kit to remove RNA. In this set of experiments, human mixed Mullerian tumor (LN1) cells grown on the International Space Station during Expedition 3 were examined for antigenic stability after removal of RNA. The cells were stored for three months in RNAlater(TradeMark) and RNA was extracted. The RNA filtrate Containing the protein was precipitated, washed, and suspended in buffer containing sodium dodecyl sulfate (SDS). Samples containing equal concentrations of protein were loaded onto SDS-polyacrylamide gels. Proteins were separated by electrophoresis and transferred by Western blot to polyvinylidene fluoride (PVDF) membrane. The Western blots were stained with an enhanced chemiluminescent ECL(Registered TradeMark)Plus detection kit (Amersham) and scanned using a Storm 840 gel image analyzer (Amersham, Molecular Dynamics). ImageQuant(Registered TradeMark)a software was used to quantify the densities of the protein bands. The ground control and flight LN1 cell samples showed a similar staining pattern over time with antibodies to vimentin, glyceraldehyde-3-phosphate dehydrogenase, and epithelial membrane antigens.

Hammond, Dianne K.↗
NASA NTRS1996

Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli

MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.

Non-NASA Center↗
DOE OSTI2021

The UPR regulator IRE1 promotes balanced organ development by restricting TOR-dependent control of cellular differentiation in Arabidopsis

Proteostasis of the endoplasmic reticulum (ER) is controlled by sophisticated signaling pathways that are collectively called the unfolded protein response (UPR) and are initiated by specialized ER membrane-associated sensors. The evidence that complete loss-of-function mutations of the most conserved of the UPR sensors, inositol-requiring enzyme 1 (IRE1), dysregulates tissue growth and development in metazoans and plants raises the fundamental question as to how IRE1 is connected to organismal growth. To address this question, we interrogated the Arabidopsis primary root, an established model for organ development, using the tractable Arabidopsis IRE1 mutant ire1a ire1b, which has marked root development defects in the absence of exogenous stress. We demonstrate that IRE1 is required to reach maximum rates of cell elongation and root growth. We also established that in the actively growing ire1a ire1b mutant root tips the Target of Rapamycin (TOR) kinase, a widely conserved pro-growth regulator, is hyperactive, and that, unlike cell proliferation, the rate of cell differentiation is enhanced in ire1a ire1b in a TOR-dependent manner. By functionally connecting two essential growth regulators, these results underpin a novel and critical role of IRE1 in organ development and indicate that, as cells exit an undifferentiated state, IRE1 is required to monitor TOR activity to balance cell expansion and maturation during organ biogenesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Structural and functional analyses of SARS-CoV-2 Nsp3 and its specific interactions with the 5’ UTR of the viral genome

ABSTRACT Non-structural protein 3 (Nsp3) is the largest open reading frame encoded in the SARS-CoV-2 genome, essential for the formation of double-membrane vesicles (DMV) wherein viral RNA replication occurs. We conducted an extensive structure-function analysis of Nsp3 and determined the crystal structures of the ubiquitin-like 1 (Ubl1), nucleic acid binding (NAB), β-coronavirus-specific marker (βSM) domains, and a sub-region of the Y domain of this protein. We show that the Ubl1, ADP-ribose phosphatase (ADRP), human SARS Unique (HSUD), NAB, and Y domains of Nsp3 bind the 5’ UTR of the viral genome and that the Ubl1 and Y domains possess affinity for recognition of this region, suggesting high specificity. The Ubl1-Nucleocapsid (N) protein complex binds the 5’ UTR with greater affinity than the individual proteins alone. Our results suggest that multiple domains of Nsp3, particularly Ubl1 and Y, shepherd the 5’ UTR of the viral genome during translocation through the DMV membrane, priming the Ubl1 domain to load the genome onto N protein. IMPORTANCE The largest protein encoded by the SARS-CoV-2 genome is Nsp3. In infected cells, this multi-domain protein forms a pore structure in the virus-induced double-membrane vesicles (DMV). We have incomplete data on Nsp3 molecular structure, and here, we describe crystal structures for multiple domains of Nsp3. It is thought that newly replicated viral RNA transits through the DMV pore; however, we possess incomplete data on which regions of Nsp3 actually interact with RNA. Here, we present data showing that five domains of Nsp3 interact with the 5’ UTR of the SARS-CoV-2 RNA, including the Y domain for which no function has ever been discovered. These data suggest that the pore structure plays an active role in recognizing the terminal end of the genome, transiting and loading the viral RNA onto the cytoplasmic nucleocapsid protein. These data help expand our knowledge of Nsp3 structure and function and the SARS-CoV-2 replication cycle.

Microbiology↗
DOE OSTI2024

VPS26 Moonlights as a β-Arrestin-like Adapter for a 7-Transmembrane RGS Protein in Arabidopsis thaliana

Extracellular signals perceived by 7-transmembrane (7TM)-spanning receptors initiate desensitization that involves the removal of these receptors from the plasma membrane. Agonist binding often evokes phosphorylation in the flexible C-terminal region and/or intracellular loop 3 of many 7TM G-protein-coupled receptors in animal cells, which consequently recruits a cytoplasmic intermediate adaptor, β-arrestin, resulting in clathrin-mediated endocytosis (CME) and downstream signaling such as transcriptional changes. Some 7TM receptors undergo CME without recruiting β-arrestin, but it is not clear how. Arrestins are not encoded in the Arabidopsis thaliana genome, yet Arabidopsis cells have a well-characterized signal-induced CME of a 7TM protein, designated Regulator of G Signaling 1 (AtRGS1). Here we show that a component of the retromer complex, Vacuolar Protein Sorting-Associated 26 (VPS26), binds the phosphorylated C-terminal region of AtRGS1 as a VPS26A/B heterodimer to form a complex that is required for downstream signaling. We propose that VPS26 moonlights as an arrestin-like adaptor in the CME of AtRGS1.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
NASA NTRS1986

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

Knisley, Keith A.↗
NASA NTRS2003

Microencapsulated Bioactive Agents and Method of Making

The invention is directed to microcapsules encapsulating an aqueous solution of a protein, drug or other bioactive substance inside a semi-permeable membrane. The microcapsules are formed by interfacial coacervation where shear forces are limited to 0-100 dynes per square centimeter. The resulting uniform microcapsules can then be subjected to dewatering in order to cause the internal solution to become supersaturated with the dissolved substance. This dewatering allows controlled nucleation and crystallization of the dissolved substance. The crystal-filled microcapsules can be stored, keeping the encapsulated crystals in good condition for further direct use in x-ray crystallography or as injectable formulations of the dissolved drug, protein or other bioactive substance.

Dennis R Morrison↗
DOE OSTI2024

An alternate route for cellulose microfibril biosynthesis in plants

Similar to cellulose synthases (CESAs), cellulose synthase–like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss Physcomitrium patens to investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils in P. patens protonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca 2+ , rather than structural differences in the microfibrils they produce.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

“The structure of the Type III secretion system export gate with CdsO, an ATPase lever arm”

Type III protein secretion systems (T3SS) deliver effector proteins from the Gram-negative bacterial cytoplasm into a eukaryotic host cell through a syringe-like, multi-protein nanomachine. Cytosolic components of T3SS include a portion of the export apparatus, which traverses the inner membrane and features the opening of the secretion channel, and the sorting complex for substrate recognition and for providing the energetics required for protein secretion. Two components critical for efficient effector export are the export gate protein and the ATPase, which are proposed to be linked by the central stalk protein of the ATPase. We present the structure of the soluble export gate homo-nonamer, CdsV, in complex with the central stalk protein, CdsO, of its cognate ATPase, both derived from Chlamydia pneumoniae. This structure defines the interface between these essential T3S proteins and reveals that CdsO engages the periphery of the export gate that may allow the ATPase to catalyze an opening between export gate subunits to allow cargo to enter the export apparatus. We also demonstrate through structure-based mutagenesis of the homologous export gate in Pseudomonas aeruginosa that mutation of this interface disrupts effector secretion. These results provide novel insights into the molecular mechanisms governing active substrate recognition and translocation through a T3SS.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

VPS45 is required for both diffuse and tip growth of Arabidopsis thaliana cells

Introduction VPS45 belongs to the Sec1/Munc18 family of proteins, which interact with and regulate Qa-SNARE function during membrane fusion. We have shown previously that Arabidopsis thaliana VPS45 interacts with the SYP61/SYP41/VTI12 SNARE complex, which locates on the trans -Golgi network (TGN). It is required for SYP41 stability, and it functions in cargo trafficking to the vacuole and in cell expansion. It is also required for correct auxin distribution during gravitropism and lateral root growth. Results As vps45 knockout mutation is lethal in Arabidopsis, we identified a mutant, vps45-3 , with a point mutation in the VPS45 gene causing a serine 284-to-phenylalanine substitution. The VPS45-3 protein is stable and maintains interaction with SYP61 and SYP41. However, vps45-3 plants display severe growth defects with significantly reduced organ and cell size, similar to vps45 RNAi transgenic lines that have reduced VPS45 protein levels. Root hair and pollen tube elongation, both processes of tip growth, are highly compromised in vps45-3 . Mutant root hairs are shorter and thicker than those of wild-type plants, and are wavy. These root hairs have vacuolar defects, containing many small vacuoles, compared with WT root hairs with a single large vacuole occupying much of the cell volume. Pollen tubes were also significantly shorter in vps45-3 compared to WT. Discussion We thus show that VPS45 is essential for proper tip growth and propose that the observed vacuolar defects lead to loss of the turgor pressure needed for tip growth.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Computational design of potent and selective binders of BAK and BAX

Potent and selective binders of the key proapoptotic proteins BAK and BAX have not been described. We use computational protein design to generate high affinity binders of BAK and BAX with greater than 100-fold specificity for their target. Both binders activate their targets when at low concentration, driving pore formation, but inhibit membrane permeabilization when in excess. Crystallography shows that the BAK binder induces BAK unfolding, exposing the α6 helix and BH3 domain. Together, these data suggest that upon binding, BAK or BAX unfold; at high binder concentrations, self-association of the partially folded BAK or BAX proteins is blocked and the membrane remains intact, whereas at low concentrations, dimers form, and the membrane ruptures. Our designed binders modulate apoptosis via direct, specific interactions with BAK and BAX and reveal that for therapeutic strategies targeting BAK and BAX, inhibition requires saturating binder concentrations at the site of action.

Berger, Stephanie↗
DOE OSTI2021

Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli , Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E . coli and B . subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E . coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E . coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Measuring the dynamic response of the thylakoid architecture in plant leaves by electron microscopy

The performance of the photosynthesis machinery in plants, including light harvesting, electron transport, and protein repair is controlled by structural changes of the thylakoid membrane system inside the chloroplasts. In particular, the structure of the stacked grana area of thylakoid membranes is highly dynamic, changing in response to different environmental cues such as light intensity. For example, the aqueous thylakoid lumen enclosed by thylakoid membranes in grana has been documented to swell in the presence of light. However, light-induced alteration of the stromal gap in the stacked grana (partition gap) and of the unstacked stroma lamellae have not been well characterized. Light-induced changes of the entire thylakoid membrane system including the lumen in both stacked and unstacked domains as well as the partition gap are presented here, and the functional implications are discussed. This structural analysis was made possible by development of a robust semi-automated image analysis method combined with optimized plant tissue fixation techniques for transmission electron microscopy generating quantitative structural results for the analysis of thylakoid ultrastructure.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

How volatile isoprenoids improve plant thermotolerance

Volatile isoprenoids mainly include isoprene and monoterpenes, which improve the thermotolerance of the emitting plant by lowering reactive oxygen species (ROS) levels, preserving chloroplast membrane ultrastructure, maintaining photosynthesis and primary metabolism, inducing heat shock proteins, and preserving growth and development. Recent data showed that isoprenoids can act as signaling molecules to improve plant thermotolerance by altering related gene expression through Ca 2+ -mediated signaling pathways. Here, to promote further understanding of isoprenoid-mediated thermotolerance mechanisms, we review current understanding of isoprenoid-induced plant thermotolerance, along with new findings describing the corresponding underlying mechanisms and putative signaling pathways. This information is beneficial for the potential utilization of isoprenoids for enhancing crop tolerance to global warming either by enhancing the emission of isoprenoids or by using isoprenoid-inspired anti-high temperature agents.

Ca2+ signal↗