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At least 415 records · Page 23
All sourcesNASA NTRSDOE OSTI
NASA NTRS2000

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

Non-NASA Center↗
DOE OSTI2023

Towards resolving protein structures at the atomic scale using atom probe tomography

In the field of Structural Biology, Atom Probe Tomography (APT) is in the nascent stages of development wherein coarse-grained visuals of proteins have been captured. The characterization of organic samples or biomolecules through the technique is currently limited to the detection of a few dominant signatures. The problem of indecipherable characterization can inherently be traced back to multiple forms of technique-specific responses to organic samples and consequent triggers leading to organic-sample and sample-medium interactions. While it is possible for captured manifestations of protein reconstructions to seemingly appear intact from a basic visual purview, the parameter-protein associative responses throughout the structure as a direct consequence of the inherent workings of the technique (until harmonized with organic sample complexity and behavior) and field evaporation-based factors make non-aberrative atomic associations infeasible. The work focused on identifying and theorizing the (above stated and other) fundamental mechanisms that stronghold the study of intricate atomic to higher order associations in proteins through APT. Attempts at structure elucidation of the cryogenic sample under study, through indirect associations (and methods) based on other imaging techniques, further revealed the distinct and highly distortive nature at the atomic scale deterring structural tunability and thus characterization of APT based cryogenic samples under analysis. As a direct counter to the atomic scale characterization problem, by taking the experiment-specific uncertainties, and probable APT-centric organic sample-based variabilities into account, a basic result is extracted and presented. Through mass-spectrometric and computational analysis, specific individual amino acids (Sulfur-containing protein-bound amino acids) in proteins and aspects of protein structure (probable backbone fragments, partial sequence - partial backbone portions) have been identified and characterized. Under analysis considerations, a few of the simplest known and easily inferable segments that favor structural deteriorations in the reconstructions are stated. Additionally, to overcome technique-specific deterrents to the characterization of biomolecules in cryogenic sample medium, the development of a protein-labeling strategy tailored to APT is suggested.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Conformational Changes of RORγ During Response Element Recognition and Coregulator Engagement

The retinoic acid receptor-related orphan receptor γ (RORγ) is a ligand-dependent transcription factor of the nuclear receptor super family that underpins metabolic activity, immune function, and cancer progression. Despite being a valuable drug target in health and disease, our understanding of the ligand-dependent activities of RORγ is far from complete. Like most nuclear receptors, RORγ must recruit coregulatory protein to enact the RORγ target gene program. To date, a majority of structural studies have been focused exclusively on the RORγ ligand-binding domain and the ligand-dependent recruitment of small peptide segments of coregulators. Herein, we examine the ligand-dependent assembly of full length RORγ:coregulator complexes on cognate DNA response elements using structural proteomics and small angle x-ray scattering. The results from our studies suggest that RORγ becomes elongated upon DNA recognition, preventing long range interdomain crosstalk. We also determined that the DNA binding domain adopts a sequence-specific conformation, and that coregulatory protein may be able to ‘sense’ the ligand- and DNA-bound status of RORγ. We propose a model where ligand-dependent coregulator recruitment may be influenced by the sequence of the DNA to which RORγ is bound. Overall, the efforts described herein will illuminate important aspects of full length RORγ and monomeric orphan nuclear receptor target gene regulation through DNA-dependent conformational changes.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1999

Transcription in archaea

Using the sequences of all the known transcription-associated proteins from Bacteria and Eucarya (a total of 4,147), we have identified their homologous counterparts in the four complete archaeal genomes. Through extensive sequence comparisons, we establish the presence of 280 predicted transcription factors or transcription-associated proteins in the four archaeal genomes, of which 168 have homologs only in Bacteria, 51 have homologs only in Eucarya, and the remaining 61 have homologs in both phylogenetic domains. Although bacterial and eukaryotic transcription have very few factors in common, each exclusively shares a significantly greater number with the Archaea, especially the Bacteria. This last fact contrasts with the obvious close relationship between the archaeal and eukaryotic transcription mechanisms per se, and in particular, basic transcription initiation. We interpret these results to mean that the archaeal transcription system has retained more ancestral characteristics than have the transcription mechanisms in either of the other two domains.

NASA Discipline Exobiology↗
NASA NTRSDate not supplied

Engineering 6-Phosphogluconate Improves Grain Yield in Heat-Stressed Maize

Endosperm starch synthesis is a primary determinant of grain yield and is sensitive to high-temperature stress. The maize chloroplast-localized 6-phosphogluconate dehydrogenase (6PGDH), PGD3, is critical for endosperm starch accumulation. Maize also has two cytosolic isozymes, PGD1 and PGD2, that are not required for kernel development. We found that cytosolic PGD1 and PGD2 isozymes have heat-stable activity, while amyloplast-localized PGD3 activity is labile under heat stress conditions. We targeted heat-stable 6PGDH to endosperm amyloplasts by fusing the Waxy1 chloroplast targeting the peptide coding sequence to the Pgd1 and Pgd2 open reading frames (ORFs). These WPGD1 and WPGD2 fusion proteins import into isolated chloroplasts, demonstrating a functional targeting sequence. Transgenic maize plants expressing WPGD1 and WPGD2 with an endosperm-specific promoter increased 6PGDH activity with enhanced heat stability in vitro. WPGD1 and WPGD2 transgenes complement the pgd3-defective kernel phenotype, indicating the fusion proteins are targeted to the amyloplast. In the field, the WPGD1 and WPGD2 transgenes can mitigate grain yield losses in high-nighttime-temperature conditions by increasing kernel number. These results provide insight into the subcellular distribution of metabolic activities in the endosperm and suggest the amyloplast pentose phosphate pathway is a heat-sensitive step in maize kernel metabolism that contributes to yield loss during heat stress.

Synthetic Biology↗
NASA NTRS1999

Mutations in a new Arabidopsis cyclophilin disrupt its interaction with protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

NASA Discipline Plant Biology↗
DOE OSTI2024

Multi-omics Resources for Understanding Gene Regulation in Response to ER Stress in Plants

Proteotoxic stress of the endoplasmic reticulum (ER) is a potentially lethal condition that ensues when the biosynthetic capacity of the ER is overwhelmed. A sophisticated and largely conserved signaling, known as the unfolded protein response (UPR), is designed to monitor and alleviate ER stress. In plants, the emerging picture of gene regulation by the UPR now appears to be more complex than ever before, requiring multi-omics-enabled network-level approaches to be untangled. In the past decade, with an increasing access and decreasing costs of next-generation sequencing (NGS) and high-throughput protein–DNA interaction (PDI) screening technologies, multitudes of global molecular measurements, known as omics, have been generated and analyzed by the research community to investigate the complex gene regulation of plant UPR. In this chapter, we present a comprehensive catalog of omics resources at different molecular levels (transcriptomes, protein–DNA interactomes, and networks) along with the introduction of key concepts in experimental and computational tools in data generation and analyses. Finally, this chapter will serve as a starting point for both experimentalists and bioinformaticians to explore diverse omics datasets for their biological questions in the plant UPR, with likely applications also in other species for conserved mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Discovery of photosynthesis genes through whole-genome sequencing of acetate-requiring mutants of Chlamydomonas reinhardtii

Large-scale mutant libraries have been indispensable for genetic studies, and the development of next-generation genome sequencing technologies has greatly advanced efforts to analyze mutants. In this work, we sequenced the genomes of 660 Chlamydomonas reinhardtii acetate-requiring mutants, part of a larger photosynthesis mutant collection previously generated by insertional mutagenesis with a linearized plasmid. We identified 554 insertion events from 509 mutants by mapping the plasmid insertion sites through paired-end sequences, in which one end aligned to the plasmid and the other to a chromosomal location. Nearly all (96%) of the events were associated with deletions, duplications, or more complex rearrangements of genomic DNA at the sites of plasmid insertion, and together with deletions that were unassociated with a plasmid insertion, 1470 genes were identified to be affected. Functional annotations of these genes were enriched in those related to photosynthesis, signaling, and tetrapyrrole synthesis as would be expected from a library enriched for photosynthesis mutants. Systematic manual analysis of the disrupted genes for each mutant generated a list of 253 higher-confidence candidate photosynthesis genes, and we experimentally validated two genes that are essential for photoautotrophic growth, CrLPA3 and CrPSBP4 . The inventory of candidate genes includes 53 genes from a phylogenomically defined set of conserved genes in green algae and plants. Altogether, 70 candidate genes encode proteins with previously characterized functions in photosynthesis in Chlamydomonas , land plants, and/or cyanobacteria; 14 genes encode proteins previously shown to have functions unrelated to photosynthesis. Among the remaining 169 uncharacterized genes, 38 genes encode proteins without any functional annotation, signifying that our results connect a function related to photosynthesis to these previously unknown proteins. This mutant library, with genome sequences that reveal the molecular extent of the chromosomal lesions and resulting higher-confidence candidate genes, will aid in advancing gene discovery and protein functional analysis in photosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic 5 engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas 6 genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, in this paper we report 7 peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand 8 DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on 9 target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much 10 smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, 11 demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

RNAi-Mediated Manipulation of Cuticle Coloration Genes in Lygus hesperus Knight (Hemiptera: Miridae)

Cuticle coloration in insects is a consequence of the accumulation of pigments in a species-specific pattern. Numerous genes are involved in regulating the underlying processes of melanization and sclerotization, and their manipulation can be used to create externally visible markers of successful gene editing. To clarify the roles for many of these genes and examine their suitability as phenotypic markers in Lygus hesperus Knight (western tarnished plant bug), transcriptomic data were screened for sequences exhibiting homology with the Drosophila melanogaster proteins. Complete open reading frames encoding putative homologs for six genes (aaNAT, black, ebony, pale, tan, and yellow) were identified, with two variants for black. Sequence and phylogenetic analyses supported preliminary annotations as cuticle pigmentation genes. In accord with observable difference in color patterning, expression varied for each gene by developmental stage, adult age, body part, and sex. Knockdown by injection of dsRNA for each gene produced varied effects in adults, ranging from the non-detectable (black 1, yellow), to moderate decreases (pale, tan) and increases (black 2, ebony) in darkness, to extreme melanization (aaNAT). Based solely on its expression profile and highly visible phenotype, aaNAT appears to be the best marker for tracking transgenic L. hesperus.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

CRISPR–Cas9 bends and twists DNA to read its sequence

In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9–RNA–DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 ‘reads’ snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction

Controlling the selectivity of a reaction is critical for target-oriented synthesis. Accessing complementary selectivity profiles enables divergent synthetic strategies, but is challenging to achieve in biocatalytic reactions given enzymes’ innate preferences of a single selectivity. Thus, it is critical to understand the structural features that control selectivity in biocatalytic reactions to achieve tunable selectivity. Here, we investigate the structural features that control the stereoselectivity in an oxidative dearomatization reaction that is key to making azaphilone natural products. Crystal structures of enantiocomplementary biocatalysts guided the development of multiple hypotheses centered on the structural features that control the stereochemical outcome of the reaction; however, in many cases, direct substitutions of active site residues in natural proteins led to inactive enzymes. Ancestral sequence reconstruction (ASR) and resurrection were employed as an alternative strategy to probe the impact of each residue on the stereochemical outcome of the dearomatization reaction. These studies suggest that two mechanisms are active in controlling the stereochemical outcome of the oxidative dearomatization reaction: one involving multiple active site residues in AzaH and the other dominated by a single Phe to Tyr switch in TropB and AfoD. Moreover, this study suggests that the flavin-dependent monooxygenases (FDMOs) adopt simple and flexible strategies to control stereoselectivity, which has led to stereocomplementary azaphilone natural products produced by fungi. This paradigm of combining ASR and resurrection with mutational and computational studies showcases sets of tools for understanding enzyme mechanisms and provides a solid foundation for future protein engineering efforts.

Science & Technology - Other Topics↗
DOE OSTI2021

Data from PNA-Assisted DNAzymes to Cleave Double-Stranded DNA for Genetic Engineering with High Sequence Fidelity

DNAzymes have been widely used in many sensing and imaging applications but have rarely been used for genetic engineering since their discovery in 1994, because their substrate scope is mostly limited to single-stranded DNA or RNA, whereas genetic information is stored mostly in double-stranded DNA (dsDNA). To overcome this major limitation, we herein report peptide nucleic acid (PNA)-assisted double-stranded DNA nicking by DNAzymes (PANDA) as the first example to expand DNAzyme activity toward dsDNA. We show that PANDA is programmable in efficiently nicking or causing double strand breaks on target dsDNA, which mimics protein nucleases and can act as restriction enzymes in molecular cloning. In addition to being much smaller than protein enzymes, PANDA has a higher sequence fidelity compared with CRISPR/Cas under the condition we tested, demonstrating its potential as a novel alternative tool for genetic engineering and other biochemical applications.

Conversion↗
DOE OSTI2024

Interactive tools for functional annotation of bacterial genomes

Automated annotations of protein functions are error-prone because of our lack of knowledge of protein functions. For example, it is often impossible to predict the correct substrate for an enzyme or a transporter. Furthermore, much of the knowledge that we do have about the functions of proteins is missing from the underlying databases. We discuss how to use interactive tools to quickly find different kinds of information relevant to a protein’s function. Many of these tools are available via PaperBLAST (http://papers.genomics.lbl.gov). Combining these tools often allows us to infer a protein’s function. Ideally, accurate annotations would allow us to predict a bacterium’s capabilities from its genome sequence, but in practice, this remains challenging. We describe interactive tools that infer potential capabilities from a genome sequence or that search a genome to find proteins that might perform a specific function of interest.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Class 2 CRISPR/Cas compositions and methods of use

Provided are compositions and methods that include one or more of: (1) a Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the effector protein, and/or a modified host cell comprising the effector protein (and/or a nucleic acid encoding the same); (2) a CRISPR/Cas guide RNA that binds to and provides sequence specificity to the Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the CRISPR/Cas guide RNA, and/or a modified host cell comprising the CRISPR/Cas guide RNA (and/or a nucleic acid encoding the same); and (3) a CRISPR/Cas transactivating noncoding RNA (trancRNA), a nucleic acid encoding the CRISPR/Cas trancRNA, and/or a modified host cell comprising the CRISPR/Cas trancRNA (and/or a nucleic acid encoding the same).

Doudna, Jennifer A.↗
NASA NTRS1984

Self-sequencing of amino acids and origins of polyfunctional protocells

The role of proteins in the origin of living things is discussed. It has been experimentally established that amino acids can sequence themselves under simulated geological conditions with highly nonrandom products which accordingly contain diverse information. Multiple copies of each type of macromolecule are formed, resulting in greater power for any protoenzymic molecule than would accrue from a single copy of each type. Thermal proteins are readily incorporated into laboratory protocells. The experimental evidence for original polyfunctional protocells is discussed.

Fox, S. W.↗
DOE OSTI2025

Amino Acid Sequence Controls Enhanced Electron Transport in Heme-Binding Peptide Monolayers

Metal-binding proteins have the exceptional ability to facilitate long-range electron transport in nature. Despite recent progress, the sequence-structure–function relationships governing electron transport in heme-binding peptides and protein assemblies are not yet fully understood. In this work, the electronic properties of a series of heme-binding peptides inspired by cytochrome bc1 are studied using a combination of molecular electronics experiments, molecular modeling, and simulation. Self-assembled monolayers (SAMs) are prepared using sequence-defined heme-binding peptides capable of forming helical secondary structures. Following monolayer formation, the structural properties and chemical composition of assembled peptides are determined using atomic force microscopy and X-ray photoelectron spectroscopy, and the electronic properties (current density–voltage response) are characterized using a soft contact liquid metal electrode method based on eutectic gallium–indium alloys (EGaIn). Our results show a substantial 1000-fold increase in current density across SAM junctions upon addition of heme compared to identical peptide sequences in the absence of heme, while maintaining a constant junction thickness. These findings show that amino acid composition and sequence directly control enhancements in electron transport in heme-binding peptides. Overall, this study demonstrates the potential of using sequence-defined synthetic peptides inspired by nature as functional bioelectronic materials.

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

Auto_PDI

Protein-DNA Interaction Workflow (PDI Workflow), a pipeline that focuses on generating high-quality docking and molecular dynamics simulations for Protein-DNA complexes. This allows us to take DNA sequences with unknown tertiary structures, accurately predict their structure, dock them with the desired target protein, and then simulate their interactions using molecular dynamics simulations

Kumar, Neeraj↗