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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Sequence-Controlled Secondary Structures and Stimuli Responsiveness of Bioinspired Polyampholytes

In this work, a comprehensive study focusing on the influence of the sequence charge pattern on the secondary structure preferences of annealed polyampholytes and their responsiveness to external stimuli is presented. Two sequences are designed composed entirely of ionizable amino acids (charge fraction f=1), and an equal number of positive and negative charges (f + =f - =0.5) with distinct charge-patterns consisting of lysine and glutamic acid monomers. The study reveals that the sequence charge pattern has a significant influence on the secondary structure preferences of polyampholytes at physiological pH. Furthermore, it shows that external stimuli such as pH, ionic strength and solvent dielectric constant can be used to modulate the secondary structure of the two designed sequences. The observed secondary structure transformations for the two sequences are also substantially different from those determined for uniformly charged polypeptides-homo-polypeptides under matching conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Aminobenzoic Acid Derivatives Obstruct Induced Fit in the Catalytic Center of the Ribosome

The Escherichia coli (E. coli) ribosome can incorporate a variety of non-l-α-amino acid monomers into polypeptide chains in vitro but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives–3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)–the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high-resolution cryo-EM structures of the ribosome with each of these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A-site). The structures reveal how the aromatic ring of each monomer sterically blocks the positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the bound water network that is believed to facilitate formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a mechanistic rationale for differences in reactivity of aminobenzoic acid derivatives relative to l-α-amino acids and each other and identify stereochemical constraints on the size and geometry of non-monomers that can be accepted efficiently by wild-type ribosomes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis , as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cryo-EM structures of light-harvesting 2 complexes from Rhodopseudomonas palustris reveal the molecular origin of absorption tuning

The genomes of some purple photosynthetic bacteria contain a multigene puc family encoding a series of α- and β-polypeptides that together form a heterogeneous antenna of light-harvesting 2 (LH2) complexes. To unravel this complexity, we generated four sets of puc deletion mutants in Rhodopseudomonas palustris , each encoding a single type of pucBA gene pair and enabling the purification of complexes designated as PucA-LH2, PucB-LH2, PucD-LH2, and PucE-LH2. The structures of all four purified LH2 complexes were determined by cryogenic electron microscopy (cryo-EM) at resolutions ranging from 2.7 to 3.6 Å. Uniquely, each of these complexes contains a hitherto unknown polypeptide, γ, that forms an extended undulating ribbon that lies in the plane of the membrane and that encloses six of the nine LH2 αβ-subunits. The γ-subunit, which is located near to the cytoplasmic side of the complex, breaks the C9 symmetry of the LH2 complex and binds six extra bacteriochlorophylls (BChls) that enhance the 800-nm absorption of each complex. The structures show that all four complexes have two complete rings of BChls, conferring absorption bands centered at 800 and 850 nm on the PucA-LH2, PucB-LH2, and PucE-LH2 complexes, but, unusually, the PucD-LH2 antenna has only a single strong near-infared (NIR) absorption peak at 803 nm. Comparison of the cryo-EM structures of these LH2 complexes reveals altered patterns of hydrogen bonds between LH2 αβ-side chains and the bacteriochlorin rings, further emphasizing the major role that H bonds play in spectral tuning of bacterial antenna complexes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cyanobacterial photosystem II reaction center design in tobacco chloroplasts increases biomass in low light

Abstract The D1 polypeptide of the photosystem II (PSII) reaction center complex contains domains that regulate primary photochemical yield and charge recombination rate. Many prokaryotic oxygenic phototrophs express two or more D1 isoforms differentially in response to environmental light needs, a capability absent in flowering plants and algae. We report that tobacco (Nicotiana tabacum) plants carrying the Synechococcus (Synechococcus elongatus PCC 7942) low-light mutation (LL-E130Q) in the D1 polypeptide (NtLL) acquire the cyanobacterial photochemical phenotype: faster photodamage in high light and significantly more charge separations in productive linear electron flow in low light. This flux increase produces 16.5% more (dry) biomass under continuous low-light illumination (100 μE m−2 s−1, 24 h). This gain is offset by the predicted lower photoprotection at high light. By contrast, the introduction of the Synechococcus high-light mutation (HL-A152S) into tobacco D1 (NtHL) has slightly increased photoprotection, achieved by photochemical quenching, but no apparent impact on biomass yield compared to wild type under the tested conditions. The universal design principle of all PSII reaction centers trades off energy conversion for photoprotection in different proportions across all phototrophs and provides a useful guidance for testing in crop plants. The observed biomass advantage under continuous low light can be transferred between evolutionarily isolated lineages to benefit growth under artificial lighting conditions. However, removal of the selective marker gene was essential to observe the growth phenotype, indicating growth penalty imposed by use of the particular spectinomycin-resistance gene.

Zhang, Yuan (ORCID:0000000291785207)↗
NASA NTRS1972

A possible step in the origin of the genetic code.

It is suggested that the earliest coding polynucleotides contained a high proportion of alternating sequences of purines and pyrimidines, and that these sequences coded for polypeptides in which hydrophobic and hydrophylic amino acids alternated. Structural properties of such alternating polypeptides are discussed.

Orgel, L. E.↗
NASA NTRS1981

Experiments on the abiotic amplification of optical activity

Experiments concerning the physical mechanisms for the abiotic generation and chemical mechanisms for the amplification of optical activity in biological compounds are reviewed. Attention is given to experiments involving the determination of the differential adsorption of racemic amino acids on d- and l-quartz, the asymmetric photolysis of racemic amino acids by circularly polarized light, and the asymmetric radiolysis of solid amino acids by longitudinally polarized electrons, and the enantiomeric enrichments thus obtained are noted. Further experiments on the amplification of the chirality in the polymerization of D, L-amino acid mixtures and the hydrolysis of D-, L-, and D, L-polypeptides are discussed. It is suggested that a repetitive cycle of partial polymerization-hydrolyses may account for the abiotic genesis of optically enriched polypeptides on the primitive earth.

Bonner, W. A.↗
NASA NTRS1992

Sequences Of Amino Acids For Human Serum Albumin

Sequences of amino acids defined for use in making polypeptides one-third to one-sixth as large as parent human serum albumin molecule. Smaller, chemically stable peptides have diverse applications including service as artificial human serum and as active components of biosensors and chromatographic matrices. In applications involving production of artificial sera from new sequences, little or no concern about viral contaminants. Smaller genetically engineered polypeptides more easily expressed and produced in large quantities, making commercial isolation and production more feasible and profitable.

Carter, Daniel C.↗
NASA NTRS1991

Archaebacterial rhodopsin sequences: Implications for evolution

It was proposed over 10 years ago that the archaebacteria represent a separate kingdom which diverged very early from the eubacteria and eukaryotes. It follows that investigations of archaebacterial characteristics might reveal features of early evolution. So far, two genes, one for bacteriorhodopsin and another for halorhodopsin, both from Halobacterium halobium, have been sequenced. We cloned and sequenced the gene coding for the polypeptide of another one of these rhodopsins, a halorhodopsin in Natronobacterium pharaonis. Peptide sequencing of cyanogen bromide fragments, and immuno-reactions of the protein and synthetic peptides derived from the C-terminal gene sequence, confirmed that the open reading frame was the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences of this gene, as well as those of other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences were calculated. These indicate very considerable evolutionary distance between each pair of genes, even in the dame organism. In spite of this, three protein sequences show extensive similarities, indicating strong selective pressures.

Lanyi, J. K.↗
NASA NTRS1997

Immunolocalization of integrin-like proteins in Arabidopsis and Chara

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

Non-NASA Center↗
NASA NTRS1990

Immunological characterization of plant ornithine transcarbamylases

Pea (Pisum sativum L.) ornithine transcarbamylase (OTC) antisera were used to investigate the immunological relatedness of several plant and animal OTC enzymes. The antisera immunoprecipitated OTC activity in all monocot and dicot species tested, and sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of immunoprecipitated protein revealed monomeric proteins ranging from 35,200 to 36,800 daltons in size. Pea OTC antisera did not recognize mammalian OTC protein. OTC activity and protein levels detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblots from homogenates of green leaf, etiolated epicotyl and cotyledon, and root tissues of pea were poorly correlated. This might result from differences in amounts of enzymatically active OTC protein in the homogenates. Alternatively, the antisera may fail to recognize different isozyme forms of OTC, which have been reported for some plant species. A putative cytosolic precursor OTC (pOTC) polypeptide exhibiting and Mr = 39,500 to 40,000 daltons was immunoprecipitated from in vitro translation mixtures of total pea leaf poly(A)+ RNA. The size of the pOTC polypeptide, as compared with mature OTC monomer (36,000 daltons), suggests that a 4 kilodalton N-terminal leader sequence, like that responsible for mitochondrial targeting of the mammalian enzyme, may be involved in organellar import of the plant enzyme.

NASA Discipline Plant Biology↗
NASA NTRS1989

The diageotropica mutant of tomato lacks high specific activity auxin binding sites

Tomato plants homozygous for the diageotropica (dgt) mutation exhibit morphological and physiological abnormalities which suggest that they are unable to respond to the plant growth hormone auxin (indole-3-acetic acid). The photoaffinity auxin analog [3H]5N3-IAA specifically labels a polypeptide doublet of 40 and 42 kilodaltons in membrane preparations from stems of the parental variety, VFN8, but not from stems of plants containing the dgt mutation. In roots of the mutant plants, however, labeling is indistinguishable from that in VFN8. These data suggest that the two polypeptides are part of a physiologically important auxin receptor system, which is altered in a tissue-specific manner in the mutant.

NASA Discipline Number 29-20↗
NASA NTRS1990

Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases

Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

NASA Program Space Biology↗
NASA NTRS1995

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

NASA Discipline Neuroscience↗
NASA NTRS1992

Structural specificity of mucosal-cell transport and metabolism of peptide drugs: implication for oral peptide drug delivery

The brush border membrane of intestinal mucosal cells contains a peptide carrier system with rather broad substrate specificity and various endo- and exopeptidase activities. Small peptide (di-/tripeptide)-type drugs with or without an N-terminal alpha-amino group, including beta-lactam antibiotics and angiotensin-converting enzyme (ACE) inhibitors, are transported by the peptide transporter. Polypeptide drugs are hydrolyzed by brush border membrane proteolytic enzymes to di-/tripeptides and amino acids. Therefore, while the intestinal brush border membrane has a carrier system facilitating the absorption of di-/tripeptide drugs, it is a major barrier limiting oral availability of polypeptide drugs. In this paper, the specificity of peptide transport and metabolism in the intestinal brush border membrane is reviewed.

Review, Tutorial↗
NASA NTRS2009

Thermodynamic Approach to Enhanced Dispersion and Physical Properties in a Carbon Nanotube/Polypeptide Nanocomposite

A high molecular weight synthetic polypeptide has been designed which exhibits favorable interactions with single wall carbon nanotubes (SWCNTs). The enthalpic and entropic penalties of mixing between these two molecules are reduced due to the polypeptide's aromatic sidechains and helical secondary structure, respectively. These enhanced interactions result in a well dispersed SWCNT/Poly (L-Leucine-ran-L-Phenylalanine) nanocomposite with enhanced mechanical and electrical properties using only shear mixing and sonication. At 0.5 wt% loading of SWCNT filler, the nanocomposite exhibits simultaneous increases in the Young's modulus, failure strain, and toughness of 8%, 120%, and 144%, respectively. At one kHz, the same nanotube loading level also enhances the dielectric constant from 2.95 to 22.81, while increasing the conductivity by four orders of magnitude.

Lovell, Conrad S.↗