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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
NASA NTRS1986

Functional and evolutionary relationships between bacteriorhodopsin and halorhodopsin in the archaebacterium, halobacterium halobium

The archaebacteria occupy a unique place in phylogenetic trees constructed from analyses of sequences from key informational macromolecules, and their study continues to yield interesting ideas on the early evolution and divergence of biological forms. It is now known that the halobacteria among these species contain various retinal-proteins, resembling eukaryotic rhodopsins, but with different functions. Two of these pigments, located in the cytoplasmic membranes of the bacteria, are bacteriorhodopsin (a light-driven proton pump) and halorhodopsin (a light-driven chloride pump). Comparison of these systems is expected to reveal structure/function relationships in these simple (primitive?) energy transducing membrane components and evolutionary relationships which had produced the structural features which allow the divergent functions. Findings indicate that very different primary structures are needed for these proteins to accomplish their different functions. Indeed, analysis of partial amino acid sequences from halo-opsin shows already that few if any long segments exist which are homologous to bacterio-opsin. Either these proteins diverged a very long time ago to allow for the observed differences, or the evolutionary clock in the halobacteria runs faster than usual.

Lanyi, J. K.↗
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 OSTI2023

A Genome-Scale Atlas Reveals Complex Interplay of Transcription and Translation in an Archaeon

The scale of post-transcriptional regulation and the implications of its interplay with other forms of regulation in environmental acclimation are underexplored for organisms of the domain Archaea. Here, we have investigated the scale of post-transcriptional regulation in the extremely halophilic archaeon Halobacterium salinarum NRC-1 by integrating the transcriptome-wide locations of transcript processing sites (TPSs) and SmAP1 binding, the genome-wide locations of antisense RNAs (asRNAs), and the consequences of RNase_2099C knockout on the differential expression of all genes. This integrated analysis has discovered that 54% of all protein-coding genes in the genome of this haloarchaeon are likely targeted by multiple mechanisms for putative post-transcriptional processing and regulation, with about 20% of genes likely being regulated by combinatorial schemes involving SmAP1, asRNAs, and RNase_2099C. Comparative analysis of mRNA levels (transcriptome sequencing [RNA-Seq]) and protein levels (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry [SWATH-MS]) for 2,579 genes over four phases of batch culture growth in complex medium generated additional evidence for the conditional post-transcriptional regulation of 7% of all protein-coding genes. We demonstrate that post-transcriptional regulation may act to fine-tune specialized and rapid acclimation to stressful environments, e.g., as a switch to turn on gas vesicle biogenesis to promote vertical relocation under anoxic conditions and modulate the frequency of transposition by insertion sequence (IS) elements of the IS200/IS605, IS4, and ISH3 families. Findings from this study are provided as an atlas in a public Web resource (https://halodata.systemsbiology.net).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Comparative analysis of KRAS4a and KRAS4b splice variants reveals distinctive structural and functional properties

KRAS, the most frequently mutated oncogene in human cancer, produces two isoforms, KRAS4a and KRAS4b, through alternative splicing. These isoforms differ in exon 4, which encodes the final 15 residues of the G-domain and hypervariable regions (HVRs), vital for trafficking and membrane localization. While KRAS4b has been extensively studied, KRAS4a has been largely overlooked. Our multidisciplinary study compared the structural and functional characteristics of KRAS4a and KRAS4b, revealing distinct structural properties and thermal stability. Position 151 influences KRAS4a’s thermal stability, while position 153 affects binding to RAF1 CRD protein. Nuclear magnetic resonance analysis identified localized structural differences near sequence variations and provided a solution-state conformational ensemble. Notably,KRAS4aexhibits substantial transcript abundance in bile ducts, liver, and stomach, with transcript levels approachingKRAS4bin the colon and rectum. Functional disparities were observed in full-length KRAS variants, highlighting the impact of HVR variations on interaction with trafficking proteins and downstream effectors like RAF and PI3K within cells.

Science & Technology - Other Topics↗
DOE OSTI2020

Internal Fragments Generated by Electron Ionization Dissociation Enhance Protein Top-Down Mass Spectrometry

Top-down proteomics by mass spectrometry (MS) involves the mass measurement of an intact protein followed by subsequent activation of the protein to generate product ions. Electron-based fragmentation methods like electron capture dissociation and electron transfer dissociation are widely used for these types of analyses. Recently, electron ionization dissociation (EID), which utilizes higher energy electrons (>20 eV) has been suggested to be more efficient for top-down protein fragmentation compared to other electron-based dissociation methods. In this paper, we demonstrate that the use of EID enhances protein fragmentation and subsequent detection of protein fragments. Protein product ions can form by either single cleavage events, resulting in terminal fragments containing the C-terminus or N-terminus of the protein, or by multiple cleavage events to give rise to internal fragments that include neither the C-terminus nor the N-terminus of the protein. Conventionally, internal fragments have been disregarded, as reliable assignments of these fragments were limited. Here, we demonstrate that internal fragments generated by EID can account for ~20–40% of the mass spectral signals detected by top-down EID-MS experiments. By including internal fragments, the extent of the protein sequence that can be explained from a single tandem mass spectrum increases from ~50 to ~99% for 29 kDa carbonic anhydrase II and 8.6 kDa ubiquitin. When searching for internal fragments during data analysis, previously unassigned peaks can be readily and accurately assigned to confirm a given protein sequence and to enhance the utility of top-down protein sequencing experiments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

LevSeq: Rapid Generation of Sequence-Function Data for Directed Evolution and Machine Learning

Sequence-function data provides valuable information about the protein functional landscape but is rarely obtained during directed evolution campaigns. Here, we present Long-read every variant Sequencing (LevSeq), a pipeline that combines a dual barcoding strategy with nanopore sequencing to rapidly generate sequence-function data for entire protein-coding genes. LevSeq integrates into existing protein engineering workflows and comes with open-source software for data analysis and visualization. The pipeline facilitates data-driven protein engineering by consolidating sequence-function data to inform directed evolution and provide the requisite data for machine learning-guided protein engineering (MLPE). LevSeq enables quality control of mutagenesis libraries prior to screening, which reduces time and resource costs. Simulation studies demonstrate LevSeq’s ability to accurately detect variants under various experimental conditions. Lastly, we show LevSeq’s utility in engineering protoglobins for new-to-nature chemistry. Widespread adoption of LevSeq and sharing of the data will enhance our understanding of protein sequence-function landscapes and empower data-driven directed evolution.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Molecular and biochemical characterization of rice developed through conventional integration of nDart1-0 transposon gene

Mutations, the genetic variations in genomic sequences, play an important role in molecular biology and biotechnology. During DNA replication or meiosis, one of the mutations is transposons or jumping genes. An indigenous transposon nDart1-0 was successfully introduced into local indica cultivar Basmati-370 from transposon-tagged line viz., GR-7895 (japonica genotype) through conventional breeding technique, successive backcrossing. Plants from segregating populationsshowed variegated phenotypes were tagged as BM-37 mutants. Blast analysis of the sequence data revealed that the GTP-binding protein, located on the BAC clone OJ1781_H11 of chromosome 5, contained an insertion of DNA transposon nDart1-0. The nDart1-0 has “A” at position 254 bp, whereas nDart1 homologs have “G”, which efficiently distinguishes nDart1-0 from its homologs. The histological analysis revealed that the chloroplast of mesophyll cells in BM-37 was disrupted with reduction in size of starch granules and higher number of osmophillic plastoglobuli, which resulted in decreased chlorophyll contents and carotenoids, gas exchange parameters (Pn, g, E, Ci), and reduced expression level of genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development. Along with the rise of GTP protein, the salicylic acid (SA) and gibberellic acid (GA) and antioxidant contents(SOD) and MDA levels significantly enhanced, while, the cytokinins (CK), ascorbate peroxidase (APX), catalase (CAT), total flavanoid contents (TFC) and total phenolic contents (TPC) significantly reduced in BM-37 mutant plants as compared with WT plants. These results support the notion that GTP-binding proteins influence the process underlying chloroplast formation. Therefore, it is anticipated that to combat biotic or abiotic stress conditions, the nDart1-0 tagged mutant (BM-37) of Basmati-370 would be beneficial.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP’s propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Molecular remodeling in Populus PdKOR RNAi roots profiled using LC-MS/MS proteomics

Plant endo-β-1,4-glucanases belonging to the Glycoside Hydrolase Family 9 have functional roles in cell wall biosynthesis and remodeling via endohydrolysis of (1→4)-β-D-glucosidic linkages. Modification of cell wall chemistry via RNAi-mediated downregulation of Populus deltoides KOR1 (PdKOR), a endo-β-1,4-glucanase gene, in Populus deltoides has been shown to have functional consequences for the composition of secondary metabolome and the ability of modified roots to interact with beneficial microbes. The molecular remodeling that underlies the observed differences at metabolic, physiological, and morphological levels in roots is not well understood. Here we used a LC-MS/MS-based proteome profiling approach to survey the molecular remodeling in root tissues of PdKOR and control plants. A total of 14316 peptides were identified and these mapped to 7139 P. deltoides proteins. Based on 90% sequence identity, the measured protein accessions represent 1187 functional protein groups. Analysis of GO categories and specific individual proteins showed differential expression of proteins relevant to plant-microbe interactions, cell wall chemistry, and metabolism. The new proteome dataset serves as a useful resource for deriving new hypotheses and empirical testing pertaining to functional roles of proteins and pathways in differential priming of plant roots to interactions with microbes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Functional characterization of prokaryotic dark matter: the road so far and what lies ahead

Eight-hundred thousand to one trillion prokaryotic species may inhabit our planet. Yet, fewer than two-hundred thousand prokaryotic species have been described. This uncharted fraction of microbial diversity, and its undisclosed coding potential, is known as the “microbial dark matter” (MDM). Next-generation sequencing has allowed to collect a massive amount of genome sequence data, leading to unprecedented advances in the field of genomics. Still, harnessing new functional information from the genomes of uncultured prokaryotes is often limited by standard classification methods. These methods often rely on sequence similarity searches against reference genomes from cultured species. This hinders the discovery of unique genetic elements that are missing from the cultivated realm. It also contributes to the accumulation of prokaryotic gene products of unknown function among public sequence data repositories, highlighting the need for new approaches for sequencing data analysis and classification. Increasing evidence indicates that these proteins of unknown function might be a treasure trove of biotechnological potential. Here, we outline the challenges, opportunities, and the potential hidden within the functional dark matter (FDM) of prokaryotes. We also discuss the pitfalls surrounding molecular and computational approaches currently used to probe these uncharted waters, and discuss future opportunities for research and applications.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

Non-NASA Center↗
NASA NTRS1972

Sequence data - Magnitude and implications of some ambiguities.

A stochastic model is applied to the divergence of the horse-pig lineage from a common ansestor in terms of the alpha and beta chains of hemoglobin and fibrinopeptides. The results are compared with those based on the minimum mutation distance model of Fitch (1972). Buckwheat and cauliflower cytochrome c sequences are analyzed to demonstrate their ambiguities. A comparative analysis of evolutionary rates for various proteins of horses and pigs shows that errors of considerable magnitude are introduced by Glx and Asx ambiguities into evolutionary conclusions drawn from sequences of incompletely analyzed proteins.

Holmquist, R.↗
NASA NTRS2001

Gene Fusion: A Genome Wide Survey

As a well known fact, organisms form larger and complex multimodular (composite or chimeric) and mostly multi-functional proteins through gene fusion of two or more individual genes which have independent evolution histories and functions. We call each of these components a module. The existence of multimodular proteins may improves the efficiency in gene regulation and in cellular functions, and thus may give the host organism advantages in adaptation to environments. Analysis of all gene fusions in present-day organisms should allow us to examine the patterns of gene fusion in context with cellular functions, to trace back the evolution processes from the ancient smaller and uni-functional proteins to the present-day larger and complex multi-functional proteins, and to estimate the minimal number of ancestor proteins that existed in the last common ancestor for all life on earth. Although many multimodular proteins have been experimentally known, identification of gene fusion events systematically at genome scale had not been possible until recently when large number of completed genome sequences have been becoming available. In addition, technical difficulties for such analysis also exist due to the complexity of this biological and evolutionary process. We report from this study a new strategy to computationally identify multimodular proteins using completed genome sequences and the results surveyed from 22 organisms with the data from over 40 organisms to be presented during the meeting. Additional information is contained in the original extended abstract.

Liang, Ping↗
DOE OSTI2026

GenomeDepot: data management system for microbial comparative genomics

Summary GenomeDepot is an open-source web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of websites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, Basic Local Alignment Search Tool (BLAST) search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools. Availability and implementation GenomeDepot is open source and distributed under the GNU General Public License via GitHub (https://github.com/aekazakov/genome-depot). GenomeDepot is implemented in Python and was tested in Ubuntu Linux. Full installation instructions and documentation are available at https://aekazakov.github.io/genome-depot/. GenomeDepot demo server is freely accessible at https://iseq.lbl.gov/demogd/.

Kazakov, Alexey [Lawrence Berkeley National Labora↗
DOE OSTI2025

GenomeDepot v1.0

GenomeDepot is a web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of web-sites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, BLAST search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools.

Kazakov, Alexey [Lawrence Berkeley National Labora↗
NASA NTRS1999

Alterations of p53 in tumorigenic human bronchial epithelial cells correlate with metastatic potential

The cellular and molecular mechanisms of radiation-induced lung cancer are not known. In the present study, alterations of p53 in tumorigenic human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells induced by a single low dose of either alpha-particles or 1 GeV/nucleon (56)Fe were analyzed by PCR-single-stranded conformation polymorphism (SSCP) coupled with sequencing analysis and immunoprecipitation assay. A total of nine primary and four secondary tumor cell lines, three of which were metastatic, together with the parental BEP2D and primary human bronchial epithelial (NHBE) cells were studied. The immunoprecipitation assay showed overexpression of mutant p53 proteins in all the tumor lines but not in NHBE and BEP2D cells. PCR-SSCP and sequencing analysis found band shifts and gene mutations in all four of the secondary tumors. A G-->T transversion in codon 139 in exon 5 that replaced Lys with Asn was detected in two tumor lines. One mutation each, involving a G-->T transversion in codon 215 in exon 6 (Ser-->lle) and a G-->A transition in codon 373 in exon 8 (Arg-->His), was identified in the remaining two secondary tumors. These results suggest that p53 alterations correlate with tumorigenesis in the BEP2D cell model and that mutations in the p53 gene may be indicative of metastatic potential.

Non-NASA Center↗
DOE OSTI2021

Comparative Virulence and Genomic Analysis of Streptococcus suis Isolates

Streptococcus suis is a zoonotic bacterial swine pathogen causing substantial economic and health burdens to the pork industry. Mechanisms used by S. suis to colonize and cause disease remain unknown and vaccines and/or intervention strategies currently do not exist. Studies addressing virulence mechanisms used by S. suis have been complicated because different isolates can cause a spectrum of disease outcomes ranging from lethal systemic disease to asymptomatic carriage. The objectives of this study were to evaluate the virulence capacity of nine United States S. suis isolates following intranasal challenge in swine and then perform comparative genomic analyses to identify genomic attributes associated with swine-virulent phenotypes. No correlation was found between the capacity to cause disease in swine and the functional characteristics of genome size, serotype, sequence type (ST), or in vitro virulence-associated phenotypes. A search for orthologs found in highly virulent isolates and not found in non-virulent isolates revealed numerous predicted protein coding sequences specific to each category. While none of these predicted protein coding sequences have been previously characterized as potential virulence factors, this analysis does provide a reliable one-to-one assignment of specific genes of interest that could prove useful in future allelic replacement and/or functional genomic studies. Collectively, this report provides a framework for future allelic replacement and/or functional genomic studies investigating genetic characteristics underlying the spectrum of disease outcomes caused by S. suis isolates.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Amino acid interacting network in the receptor-binding domain of SARS-CoV-2 spike protein

The relation between amino acid (AA) sequence and biologically active conformation controls the process of polypeptide chains folding into three-dimensional (3d) protein structures. The recent achievements in the resolution achieved in cryo-electron microscopy coupled with improvements in computational methodologies have accelerated the analysis of structures and properties of proteins. However, the detailed interaction between AAs has not been fully elucidated. Herein, we present a de novo method to evaluate inter-amino acid interactions based on the concept of accurately evaluating the amino acid bond pairs (AABP). The results obtained enabled the identification of complex 3d long-range interconnected AA interacting network in proteins. The method is applied to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. We show that although nearest-neighbor AAs in the primary sequence have large AABP, other nonlocal AAs make substantial contribution to AABP with significant participation of both covalent and hydrogen bonding. Detailed analysis of AABP in RBD reveals the pivotal role they play in sequence conservation with profound implications on residue mutations and for therapeutic drug design. This approach could be easily applied to many other proteins of biomedical interest in life sciences.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗