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At least 883 records · Page 49

Characterization of photosystem 1 chlorophyll a/b-binding apoprotein accumulation in developing soybean using type-specific antibodies

The structure and supramolecular assembly of the soybean photosystem 1 (PS 1) chlorophyll a/b-binding antenna (LHC 1) was examined. We identified the subunit composition of LHC 1 in soybean and followed the accumulation of individual subunits during light-induced assembly. We observed four LHC 1 subunits, at 23, 22, 21 and 20.5 kDa, obtained partial sequence information by amino-terminal sequence analysis, and classified the 20.5, 22, and 21 kDa subunits as being encoded by type I, II, and IV chlorophyll a/b binding protein genes, respectively. Antisera against LHC 1 subunits were used to follow the accumulation of individual subunits during the light-initiated transition from etioplast to chloroplast. Several points are noteworthy. First, monospecific antibody against the 22 kDa subunit decorated a 25 kDa peptide in etiolated tissue, which declined during maturation. This decline correlated with the light-induced appearance of mature 22 kDa peptide, suggesting a precursor/product relationship. Second, the same antibody identified a 22 kDa protein in mature corn, but not a larger band in etiolated corn, suggesting that LHC 1 accumulation is regulated differently between species before the onset of chlorophyll biosynthesis. Third, the mature 22 kDa subunit appeared somewhat later than the other LHC 1 peptides during greening, implying that this subunit is less intimately associated with the PS1 core than are the subunits appearing earlier in development.

Non-NASA Center↗

Left-handed Z-DNA: structure and function

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Review↗

Polar algae flaunt their zinc assets

Metal ions have been at the centre of pivotal points in the evolution of extant life. Oxygen-evolving photosynthesis, which irrevocably reshaped the geochemistry and biology of our planet, has an absolute requirement for metal ions to harvest light, split water and transfer electrons. Access to abundant oxygen then led to the propagation of organisms capable of oxidative metabolism, a process that is also dependent on metal ions for electron transfer and reduction of molecular oxygen. Because of the essential links between metal ions and the proteins that require them to function, as well as changes in metal bioavailability through time, metals have shaped the trajectories that evolution can take. Iron typically steals this show, but with access to whole-genome sequences and transcriptomes, the imprint that zinc has made on biology is coming into focus. Writing in Nature Ecology & Evolution, Ye and colleagues present new insights into the complex connections between zinc bioavailability, adaptation of algae to the polar oceans and the evolution of regulatory networks. By combining field and laboratory-based analyses, the authors suggest that expanded families of zinc-binding proteins have enabled the green alga Microglena sp. YARC to flourish in the harsh conditions of the polar Southern Ocean (Fig. 1). As the waters where this alga occurs are known for their enrichment of zinc, the authors further propose that availability of this metal ion was directly responsible for successful microalgal colonization of polar oceans. To test this hypothesis, the authors compare meta-transcriptomes collected from pole-to-pole and find positive correlations between higher copy numbers for transcripts encoding putative zinc-binding proteins, higher latitudes, lower surface temperatures and dissolved zinc.

59 BASIC BIOLOGICAL SCIENCES↗

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

NASA Program NSCORT↗

Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short β-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREB TAD ) construct. The CREB TAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. In conclusion, our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

59 BASIC BIOLOGICAL SCIENCES↗

On the Rapid Calculation of Binding Affinities for Antigen and Antibody Design and Affinity Maturation Simulations

The accurate and efficient calculation of protein-protein binding affinities is an essential component in antibody and antigen design and optimization, and in computer modeling of antibody affinity maturation. Such calculations remain challenging despite advances in computer hardware and algorithms, primarily because proteins are flexible molecules, and thus, require explicit or implicit incorporation of multiple conformational states into the computational procedure. The astronomical size of the amino acid sequence space further compounds the challenge by requiring predictions to be computed within a short time so that many sequence variants can be tested. In this study, we compare three classes of methods for antibody/antigen (Ab/Ag) binding affinity calculations: (i) a method that relies on the physical separation of the Ab/Ag complex in equilibrium molecular dynamics (MD) simulations, (ii) a collection of 18 scoring functions that act on an ensemble of structures created using homology modeling software, and (iii) methods based on the molecular mechanics-generalized Born surface area (MM-GBSA) energy decomposition, in which the individual contributions of the energy terms are scaled to optimize agreement with the experiment. When applied to a set of 49 antibody mutations in two Ab/HIV gp120 complexes, all of the methods are found to have modest accuracy, with the highest Pearson correlations reaching about 0.6. In particular, the most computationally intensive method, i.e., MD simulation, did not outperform several scoring functions. The optimized energy decomposition methods provided marginally higher accuracy, but at the expense of requiring experimental data for parametrization. Within each method class, we examined the effect of the number of independent computational replicates, i.e., modeled structures or reinitialized MD simulations, on the prediction accuracy. We suggest using about ten modeled structures for scoring methods, and about five simulation replicates for MD simulations as a rule of thumb for obtaining reasonable convergence. We anticipate that our study will be a useful resource for practitioners working to incorporate binding affinity calculations within their protein design and optimization process.

59 BASIC BIOLOGICAL SCIENCES↗

Protein N -Glycans in Healthy and Sclerotic Glomeruli in Diabetic Kidney Disease

Diabetes is expected to directly affect renal glycosylation; yet to date, there has not been a comprehensive evaluation of alterations in N-glycan composition in the glomeruli of patients with diabetic kidney disease (DKD). Here, we used untargeted mass spectrometry imaging to identify N-glycan structures in healthy and sclerotic glomeruli in formalin-fixed paraffin-embedded sections from needle biopsies of five patients with DKD and three healthy kidney samples. Regional proteomics was performed on glomeruli from additional biopsies from the same patients to compare the abundances of enzymes involved in glycosylation. Secondary analysis of single-nucleus RNA sequencing (snRNAseq) data were used to inform on transcript levels of glycosylation machinery in different cell types and states. We detected 120 N-glycans, and among them, we identified 12 of these protein post-translated modifications that were significantly increased in glomeruli. All glomeruli-specific N-glycans contained an N-acetyllactosamine epitope. Five N-glycan structures were highly discriminant between sclerotic and healthy glomeruli. Sclerotic glomeruli had an additional set of glycans lacking fucose linked to their core, and they did not show tetra-antennary structures that were common in healthy glomeruli. Orthogonal omics analyses revealed lower protein abundance and lower gene expression involved in synthesizing fucosylated and branched N-glycans in sclerotic podocytes. In snRNAseq and regional proteomics analyses, we observed that genes and/or proteins involved in sialylation and N-acetyllactosamine synthesis were also downregulated in DKD glomeruli, but this alteration remained undetectable by our spatial N-glycomics assay. Integrative spatial glycomics, proteomics, and transcriptomics revealed protein N-glycosylation characteristic of sclerotic glomeruli in DKD.

60 APPLIED LIFE SCIENCES↗

Robust, versatile DNA FISH probes for chromosome-specific repeats in Caenorhabditis elegans and Pristionchus pacificus

Repetitive DNA sequences are useful targets for chromosomal fluorescence in situ hybridization. We analyzed recent genome assemblies of Caenorhabditis elegans and Pristionchus pacificus to identify tandem repeats with a unique genomic localization. Based on these findings, we designed and validated sets of oligonucleotide probes for each species targeting at least 1 locus per chromosome. These probes yielded reliable fluorescent signals in different tissues and can easily be combined with the immunolocalization of cellular proteins. Synthesis and labeling of these probes are highly cost-effective and require no hands-on labor. The methods presented here can be easily applied in other model and nonmodel organisms with a sequenced genome.

59 BASIC BIOLOGICAL SCIENCES↗

Single-cell proteomics of Arabidopsis leaf mesophyll reveals dynamic protein responses to water-deficit stress

Background The application of single-cell omics tools to biological systems can provide unique insights into diverse cellular populations and their heterogeneous responses to internal and external perturbations. Thus far, most single-cell studies in plant systems have been limited to RNA-sequencing approaches, which only provide indirect readouts of cellular functions. Results Here, we present a single-cell proteomics workflow for plant cells that integrates tape-sandwich protoplasting, piezoelectric cell sorting, nanoPOTS sample preparation, and ion mobility-based MS data acquisition method for label-free single-cell proteomics analysis of Arabidopsis leaf mesophyll cells. From a single leaf protoplast, over 3,000 proteins were quantified with high precision. The workflow is demonstrated to identify stress associated changes in protein abundance by analyzing 117 protoplasts from well-watered and water-deficit stressed plants. Additionally, we describe a new approach for constructing covarying protein networks at the single-cell level and demonstrate how single-cell protein covariation analysis can reveal previously unrecognized protein functions while also capturing stress-induced changes in protein–protein dynamics. Conclusions The label-free scProteomic approach presented here represents a significant advance through the demonstration of a facile protoplast isolation method combined with deep and precise proteomic coverage of Arabidopsis leaf mesophyll cell types. We believe this study will serve as an informative reference to future plant scProteomic investigations.

Arabidopsis↗

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

NASA Discipline Musculoskeletal↗

Investigations with methanobacteria and with evolution of the genetic code

Mycoplasma capricolum was found by Osawa et al. to use UGA as the code of tryptophan and to contain 75% A + T in its DNA. This change could have been from evolutionary pressure to replace C + G by A + T. Numerous studies have been reported of evolution of proteins as measured by amino acid replacements that are observed when homologus proteins, such as hemoglobins from various vertebrates, are compared. These replacements result from nucleotide substitutions in amino acid codons in the corresponding genes. Simultaneously, silent nucleotide substitutions take place that can be studied when sequences of the genes are compared. These silent evolutionary changes take place mostly in third positions of codons. Two types of nucleotide substitutions are recognized: pyrimidine-pyrimidine and purine-purine interchanges (transitions) and pyriidine-purine interchanges (transversions). Silent transitions are favored when a corresponding transversion would produce an amino acid replacement. Conversely, silent transversions are favored by probability when transitions and transversions will both be silent. Extensive examples of these situations have been found in protein genes, and it is evident that transversions in silent positions predominate in family boxes in most of the examples studied. In associated research a streptomycete from cow manure was found to produce an extracellular enzyme capable of lysing the pseudomurein-contining methanogen Methanobacterium formicicum.

Jukes, T. H.↗

MAP kinase pathways in the yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Non-NASA Center↗

Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

Non-NASA Center↗

Hepatic transcriptome analyses of juvenile white bass ( Morone chrysops ) when fed diets where fish meal is partially or totally replaced by alternative protein sources

White bass (Morone chrysops) are a popular sportfish throughout the southern United States, and one parent of the commercially-successful hybrid striped bass (M. chrysops male x M. saxatilis female). Currently, white bass are cultured using diets formulated for other carnivorous fish, such as largemouth bass (Micropterus salmoides) or hybrid striped bass and contain a significant percentage of marine fish meal. Since there are no studies regarding the utilization of alternative proteins in this species, we evaluated the global gene expression of white bass fed diets in which fish meal was partially or totally replaced by various combinations of soybean meal, poultry by-product meal, canola meal, soy protein concentrate, wheat gluten, or a commercial protein blend (Pro Cision™). Six isonitrogenous (40% protein), isolipidic (11%), and isocaloric (17.1 kJ/g) diets were formulated to meet the known nutrient and energy requirements of largemouth bass and hybrid striped bass using nutrient availability data for most of the dietary ingredients. One of the test diets consisted exclusively of plant protein sources. Juvenile white bass (40.2 g initial weight) were stocked into a flow-through aquaculture system (three tanks/diet; 10 fish/tank) and fed the test diets twice daily to satiation for 60 days. RNA sequencing and bioinformatic analyses revealed significant differentially expressed genes between all test diets when compared to fish meal control. A total of 1,260 differentially expressed genes were identified, with major ontology relating to cell cycle and metabolic processes as well as immune gene functions. This data will be useful as a resource for future refinements to moronid diet formulation, as marine fish meal becomes limiting and plant ingredients are increasingly added as a reliable protein source

59 BASIC BIOLOGICAL SCIENCES↗

Multi-Omics Driven Metabolic Network Reconstruction and Analysis of Lignocellulosic Carbon Utilization in Rhodosporidium toruloides

An oleaginous yeast Rhodosporidium toruloides is a promising host for converting lignocellulosic biomass to bioproducts and biofuels. In this work, we performed multi-omics analysis of lignocellulosic carbon utilization in R. toruloides and reconstructed the genome-scale metabolic network of R. toruloides . High-quality metabolic network models for model organisms and orthologous protein mapping were used to build a draft metabolic network reconstruction. The reconstruction was manually curated to build a metabolic model using functional annotation and multi-omics data including transcriptomics, proteomics, metabolomics, and RB-TDNA sequencing. The multi-omics data and metabolic model were used to investigate R. toruloides metabolism including lipid accumulation and lignocellulosic carbon utilization. The developed metabolic model was validated against high-throughput growth phenotyping and gene fitness data, and further refined to resolve the inconsistencies between prediction and data. We believe that this is the most complete and accurate metabolic network model available for R. toruloides to date.

09 BIOMASS FUELS↗

De novo design of small beta barrel proteins

Small beta barrel proteins are attractive targets for computational design because of their considerable functional diversity despite their very small size (<70 amino acids). However, there are considerable challenges to designing such structures, and there has been little success thus far. Because of the small size, the hydrophobic core stabilizing the fold is necessarily very small, and the conformational strain of barrel closure can oppose folding; also intermolecular aggregation through free beta strand edges can compete with proper monomer folding. Here, we explore the de novo design of small beta barrel topologies using both Rosetta energy–based methods and deep learning approaches to design four small beta barrel folds: Src homology 3 (SH3) and oligonucleotide/oligosaccharide-binding (OB) topologies found in nature and five and six up-and-down-stranded barrels rarely if ever seen in nature. Both approaches yielded successful designs with high thermal stability and experimentally determined structures with less than 2.4 Å rmsd from the designed models. Using deep learning for backbone generation and Rosetta for sequence design yielded higher design success rates and increased structural diversity than Rosetta alone. The ability to design a large and structurally diverse set of small beta barrel proteins greatly increases the protein shape space available for designing binders to protein targets of interest.

59 BASIC BIOLOGICAL SCIENCES↗

An active site loop toggles between conformations to control antibiotic hydrolysis and inhibition potency for CTX-M β-lactamase drug-resistance enzymes

Abstract β-lactamases inactivate β-lactam antibiotics leading to drug resistance. Consequently, inhibitors of β-lactamases can combat this resistance, and the β-lactamase inhibitory protein (BLIP) is a naturally occurring inhibitor. The widespread CTX-M-14 and CTX-M-15 β-lactamases have an 83% sequence identity. In this study, we show that BLIP weakly inhibits CTX-M-14 but potently inhibits CTX-M-15. The structure of the BLIP/CTX-M-15 complex reveals that binding is associated with a conformational change of an active site loop of β-lactamase. Surprisingly, the loop structure in the complex is similar to that in a drug-resistant variant (N106S) of CTX-M-14. We hypothesized that the pre-established favorable loop conformation of the N106S mutant would facilitate binding. The N106S substitution results in a ~100- and 10-fold increase in BLIP inhibition potency for CTX-M-14 and CTX-M-15, respectively. Thus, this indicates that an active site loop in β-lactamase toggles between conformations that control antibiotic hydrolysis and inhibitor susceptibility. These findings highlight the role of accessible active site conformations in controlling enzyme activity and inhibitor susceptibility as well as the influence of mutations in selectively stabilizing discrete conformations.

59 BASIC BIOLOGICAL SCIENCES↗

Exploring life’s hidden majority: microbial dark matter symposium highlights

The Microbial Dark Matter Symposium held on August 28–29, 2025, in Laguna Beach, Orange County, CA, convened a multidisciplinary group of scientists to address the vast unknowns in microbial life—from uncultured taxa and uncharacterized proteins to elusive viruses and spacefaring microbes. Set against a scenic coastal backdrop, the symposium highlighted advances in single-cell genomics, proximity ligation sequencing, and artificial intelligence-ready bioinformatics, while also probing the limits of microbial persistence, metabolism, and ecological distribution. Sessions explored microbial dark matter from multiple dimensions: cultivability, where new strategies are enabling recovery of elusive microbes; functional ambiguity, where metagenomic dark zones are illuminated by computational annotation; and genomic representation, where single-cell methods bridge gaps left by shotgun community sequencing. Researchers shared breakthroughs in identifying atmospheric microbiomes, “dark oxygen” production in groundwater ecosystems, and microbial survival on the International Space Station. The symposium emphasized integration of methods, disciplines, and ecosystems, advancing a collective push to illuminate the microbial dark matter on Earth and beyond. By highlighting emerging tools, pressing questions, and cross-domain insights, the symposium underscored the need for collaborative, open, and adaptive approaches to study the microbial unknown. The meeting marks a pivotal moment in microbiology, where cultivating knowledge of the uncultivated promises transformative understanding of life, everywhere.

Podar, Mircea [ORNL] (ORCID:0000000327760205)↗