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At least 1,135 records · Page 63

All sources NASA NTRS DOE OSTI

DOE OSTI2016

Crystal structure of Clostridium difficile toxin A

Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA 1832 ), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics.

bacterial pathogenesis↗

DOE OSTI2017

NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

We synthesized the Hepatitis C virus (HCV) RNA by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by preformed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPO R ) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPO S ) viruses (e.g. H77S.3 and N.2). Furthermore, in luciferase assays, LPO S HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPO R HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNAdependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPO S H77S.3 and the LPO R H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. The mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPO S and LPO R viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2019

Chiral Recognition of Lipid Bilayer Membranes by Supramolecular Assemblies of Peptide Amphiphiles

On the basis of the exclusive existence of homochirality in biomolecules and the well-known phenomenon of chiral recognition, it is obvious that chirality is a crucial factor in biological events. We report here that supramolecular assemblies of peptide amphiphiles interact with lipid bilayer membranes in a stereospecific manner. When negatively charged chiral phospholipid bilayer vesicles were subjected to the assemblies, we found that peptide amphiphiles with l-amino acids show stronger affinity for the liposomes compared to the ones with d-amino acids. To examine their biological functions, we tested the cytotoxicity of nanofibers against mammalian primary cells using human bone marrow mesenchymal stem cells and murine astroglial cells. In this work, we demonstrated that cell viability increased when d-amino acids were incorporated in the structure of peptide amphiphiles, which is consistent with our finding of their weaker interactions with lipid bilayer membranes.

36 MATERIALS SCIENCE↗

DOE OSTI2013

Expanding the eukaryotic genetic code

This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

Expanding the eukaryotic genetic code

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2017

Expanding the eukaryotic genetic code

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1982

Oxidative peptide /and amide/ formation from Schiff base complexes

One hypothesis of the origin of pre-modern forms of life is that the original replicating molecules were specific polypeptides which acted as templates for the assembly of poly-Schiff bases complementary to the template, and that these polymers were then oxidized to peptide linkages, probably by photo-produced oxidants. A double cycle of such anti-parallel complementary replication would yield the original peptide polymer. If this model were valid, the Schiff base between an N-acyl alpha mino aldehyde and an amino acid should yield a dipeptide in aqueous solution in the presence of an appropriate oxidant. In the present study it is shown that the substituted dipeptide, N-acetyl-tyrosyl-tyrosine, is produced in high yield in aqueous solution at pH 9 through the action of H2O2 on the Schiff-base complex between N-acetyl-tyrosinal and tyrosine and that a great variety of N-acyl amino acids are formed from amino acids and aliphatic aldehydes under similar conditions.

Strehler, B. L.↗

NASA NTRS1988

Evolution of E. coli tRNA(Trp)

It has been shown by Lacey et al. (1985) that, in general, the hydrophobicity ranking of an amino acid correlates with that of its anticodonic nucleotide, with tryptophan being one of the four amino acids for which this rule does not apply. It was proposed that this failure to correlate was due to the fact that the anticodon assignments for the four amino acids were made late, after the mutation of existing tRNAs. In this paper, the evolution of E. coli tRNA(Trp) is examined by comparing its homology with other E. coli tRNAs. The results demonstrate the presence of an evolutionary relationship between E. coli tRNA(Trp) and tRNA(Gly) or tRNA(Arg) molecules, and support the idea of the late assignment of anticodon to Trp.

Staves, Mark P.↗

DOE OSTI2015

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ- d -Glu-A 2 pm (A 2 pm is diaminopimelic acid) cysteine amidase (or dl -endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l -Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCE Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

Xu, Qingping↗

DOE OSTI2016

Poxvirus uracil‐DNA glycosylase—An unusual member of the family I uracil‐DNA glycosylases

Abstract Uracil‐DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil‐DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non‐enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus‐specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA‐binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three‐dimensional structure of D4. An overview of the current state of the knowledge on the structure‐function relationship of D4 is provided here.

Schormann, Norbert↗

DOE OSTI2016

CO2 Reduction Catalyzed by Nitrogenase: Pathways to Formate, Carbon Monoxide, and Methane

The reduction of N2 to NH3 by Mo-dependent nitrogenase at its active-site metal cluster FeMo-cofactor utilizes reductive elimination (re) of Fe-bound hydrides with obligatory loss of H2 to activate the enzyme for binding/reduction of N2. Earlier work showed that wild type nitrogenase and a nitrogenase with amino acid substitutions in the MoFe protein near FeMo-cofactor can catalytically reduce CO2 by 2 or 8 electrons/protons to carbon monoxide (CO) and methane (CH4) at low rates. Here, it is demonstrated that nitrogenase preferentially reduces CO2 by 2 electrons/protons to formate (HCOO-) at rates >10 times higher than rates of CO2 reduction to CO and CH4. The failure of H2 to inhibit CO2 reduction rules out a re mechanism in this case. Quantum mechanical (QM) calculations on the activated FeMo-cofactor with a bound hydride (E2(2H) state) favor a direct reaction of CO2 with the hydride (‘direct hydride transfer’ reaction pathway), with facile hydride transfer to CO2 yielding formate. In contrast, a significant barrier is observed for reaction of Fe-bound CO2 with the hydride (‘associative’ reaction pathway), which leads to CO and CH4. Remarkably, in the direct hydride transfer pathway, the Fe-H behaves as a hydridic hydrogen, whereas in the associative pathway it acts as a protic hydrogen. MoFe proteins with amino acid substitutions near FeMo-cofactor (a-70Val?Ala, a -195His?Gln) are found to significantly alter the distribution of products between formate and CO/CH4. This work is based upon work supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (LCS and DRD), the Division of Chemical Sciences, Geosciences, and Bio-Sciences (SR) and the NIH (GM 111097; BMH).

Khadka, Nimesh↗

NASA NTRS2010

Formation of Nucleobases from the UV Photo-Irradiation of Pyrimidine in Astrophysical Ice Analogs

Astrochemistry laboratory simulations have shown that complex organic molecules including compounds of astrobiological interest can be formed under interstellarl/circumstellar conditions from the vacuum UV irradiation of astrophysical ice analogs containing H2O, CO, CO2, CH3OH, NH13, etc. Of all prebiotic compounds, the formation of amino acids under such experimental conditions has been the most extensively studied. Although the presence of amino acids in the interstellar medium (ISM) has yet to be confirmed, they have been detected in meteorites, indicating that biomolecules and/or their precursors can be formed under extraterrestrial, abiotic conditions. Nucleobases, the building blocks of DNA and RNA, as well as other 1V-heterocycles, have also been detected in meteorites, but like amino acids, they have yet to be observed in the ISM. In this work, we present an experimental study of the formation of pyrimidine-based compounds from the UV photo-irradiation of pyrimidine in ice mixtures containing H2O, NH3, and/or CH3OH at low temperature and pressure.

Milam, S. N.↗

NASA NTRS2001

Organic Chemistry of Carbonaceous Meteorites

Chiral and carbon-isotopic analyses of isovaline have been carried out on numerous samples of the Murchison and one sample of the Murray carbonaceous chondrite. The isovaline was found to be heterogeneous with regard to enantiomeric excess (ee) both between samples and within a single Murchison sample. L-Excesses ranging from 0 to 15% were observed. The isovaline delta(sup 13) C was found to be about +18%. No evidence was obtained suggesting terrestrial contamination in the more abundant L-enantiomer. A correlation was observed between isovaline (also alpha - aminoisobutyric acid) concentration and PCP content of five CM chondrites. It is suggested that isovaline, along with other meteoritic a-methyl amino acids with ee, are of presolar origin. The possible formation of ee in extraterrestrial amino acids by exposure to circularly polarized light or by magnetochiral photochemistry is discussed. Key words: Murchison meteorite, Murray meteorite, amino acids, isovaline, chirality, carbon isotopes, PCP.

Cronin, John R.↗

NASA NTRS1975

Looking forward to the present

The present understanding of the origin of life based on Oparin's (1924) conception of life as a manifestation of matter in a special stage of its development is outlined. The results of chemical analyses of lunar samples are discussed, and their implications regarding chemical reactions beginning with carbonaceous dust are considered. Amino acids isolated from lunar samples taken from different sites show a high degree of similarity, in agreement with the hypothesis that carbon compounds, including amino acid precursors, were implanted on the lunar surface by components of the solar wind. The amino acid precursors are most likely a cyanide compound. Overlapping amino acid/carbon ratios from lunar samples and samples of meteorites suggest a common cosmochemical state of carbon in the solar system.

Fox, S. W.↗

DOE OSTI2014

The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4 (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2003

Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90)

We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.

STS-90 Shuttle Project↗

NASA NTRS2005

Palmer Quest: A Feasible Nuclear Fission "Vision Mission" to the Mars Polar Caps

We are engaged in a NASA Vision Mission study, called Palmer Quest after the American Antarctic explorer Nathaniel Palmer, to assess the presence of life and evaluate the habitability of the basal domain of the Mars polar caps. We address this goal through four objectives: 1. Determine the presence of amino acids, nutrients, and geochemical heterogeneity in the ice sheet. 2. Quantify and characterize the provenance of the amino acids in Mars ice. 3. Assess the stratification of outcropped units for indications of habitable zones. 4. Determine the accumulation of ice, mineralogic material, and amino acids in Mars ice caps over the present epoch. Because of the defined scientific goal for the vision mission, the Palmer Quest focus is astrobiological; however, the results of the study make us optimistic that aggressive multi-platform in-situ missions that address a wide range of objectives, such as climate change, can be supported by variations of the approach used on this mission. Mission Overview: The Palmer Quest baseline

Carsey, F. D.↗

DOE OSTI2021

Mutant xylan biosynthetic enzymes capable of dominant suppression of xylan biosynthesis

The present invention provides for a polypeptide capable of dominant suppression of a first naturally occurring IRX10, wherein the polypeptide comprises an amino acid sequence having at least 70% identity as compared to a second naturally occurring IRX10 wherein the polypeptide comprises one or more of the conserved amino acid indicated in FIG. 2 substituted with a different amino acid residue.

Brandon, Andrew↗