Results for “protein labeling” — Page 20

DOE OSTI2024

National User Resource for Biological Accelerator Mass Spectrometry

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

DOE OSTI2025

National User Resource for Biological Accelerator Mass Spectrometry (Final Report)

The National User Resource for Biological Accelerator Mass Spectrometry (User Resource) will provide isotopic analysis (primarily radiocarbon or 14C) by accelerator mass spectrometry (AMS) for NIH- funded researchers across the United States and will be the only User Resource of its type in the United States. The User Resource will provide measurement capability and expertise to a research community that requires highly sensitive, quantitative isotope analyses. Since commissioning a new accelerator mass spectrometer in June 2014, we have measured over 4000 samples a year for collaborators and service users. The User Resource will enable us to continue to meet these research needs, as well as provide for new users whose research programs would benefit from AMS as a measurement tool. The User Resource’s forte will be ultra-high sensitivity quantitation of radiocarbon and selected other radioisotopes for research studies where isotopes are required. Radioisotope labeling studies have been and will continue to be an important tool for addressing many complex biomedical science problems. AMS is a specialized and unique type of mass spectrometry that provides absolute quantitation of radiocarbon and other relevant radioisotopes with extreme sensitivity, having limits of detection in real samples on the order of a few attomol/mg of sample at measurement precisions of ~3%. It is the only instrumental method capable of quantifying radioisotope-labeled agents routinely in real-world samples with such precision and sensitivity. The sensitivity of AMS allows for the quantification of radiolabeled metabolites in extremely complex matrices of cells and organisms at very low concentrations and in small samples. AMS allows studies to be conducted without perturbing metabolism leading to more relevant quantification of metabolic rates and pathways. In addition, it enables quantification of pharmacokinetic and metabolic properties of toxicants at environmentally relevant concentrations in model systems as well as the ability to quantify pharmacokinetics and other molecular endpoints directly in humans. Such quantitative assessments can 1) improve risk assessment for toxicants, 2) address safety and efficacy considerations for therapeutic entities, 3) deepen understanding of xenobiotic and intermediary metabolism, 4) help understand the interactions between critical molecular pathways, and 5) improve efforts to model and predict various metabolic and biological states. These capabilities have been applied in a number of areas including research in carcinogenesis, toxicology, nutrition, pharmacology/drug development and basic biological science. As a NIGMS National Resource the National User Resource for Biological Accelerator Mass Spectrometry will help NIH funded scientists achieve a deeper understanding of the etiology of human health concerns by (1) enabling the quantification of pharmacokinetics and other molecular endpoints directly in humans; (2) offering the ability to conduct quantitative studies using biologics such as proteins or lipids, and thereby reducing the amount of radioisotope usage in biomedical labs; and (3) enabling more relevant studies of metabolic pathways in health and disease through the use of much lower, more biologically-relevant, concentrations of metabolic substrates in cells and intact organisms. Such studies support NIGMS’s basic biomedical research areas that contribute to the understanding of fundamental cellular and physiological principles and enable research supported by the Biophysics, Biomedical Technology, and Computational Biosciences (BBCB); Genetics and Molecular, Cellular, and Developmental Biology (GMCDB); Pharmacology, Physiology, Biological Chemistry (PPBC) and Training, Workforce Development, and Diversity (TWD) Divisions. Over the next five years, our goals are to: 1. Improve the efficiency of operation for AMS measurements through installation of new interfaces to our AMS systems, technical modifications to improve gas accepting ion source efficiency and upgrading our data analysis software for improved ease of use and data reporting. 2. Increase the accessibility and visibility of ultra-sensitive 14C measurements for the biomedical research community by training of new investigators and expanding our national user base. 3. Provide high throughput, ultra-sensitive 14C analysis for the NIGMS and NIH user community.

47 OTHER INSTRUMENTATION↗

DOE OSTI2021

Elucidation of Aromatic Catabolic Pathways in White-Rot Fungi

This project aims to investigate the hypothesis that white-rot fungi can simultaneously depolymerize lignin extracellularly and catabolize depolymerization products intracellularly as carbon and energy sources. Evaluating this hypothesis will provide deeper understanding of the role of white-rot fungi in facilitating carbon sequestration in Nature. Additionally, identifying the most promising fungal strains for lignin turnover and catabolism will catalyze future efforts in genetic tool development to enable metabolic engineering in white-rot fungi for lignin bioconversion to bioproducts. Lignin is the second most abundant plant-based biopolymer on Earth and represents up to 40% of the energy density of lignocellulosic biomass. Even though lignin is a massive natural carbon and energy reservoir, only a small group of basidiomycete fungi, namely white-rot fungi (WRF), have evolved the ability to efficiently depolymerize and mineralize lignin to CO2 and H2O. Considerable research efforts have been undertaken to understand how WRF depolymerize lignin but the biochemical reactions that convert lignin into CO2 have been largely neglected. In fact, it is unclear if WRF intracellularly catabolize lignin-derived aromatic compounds to utilize them as a carbon and energy source, or rather if lignin is depolymerized and mineralized extracellularly merely to facilitate access to cellulose and hemicellulose for use as a primary carbon source. To date, we have employed 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to definitively demonstrate that two WRF, Trametes versicolor and Gelatoporia (Ceriporiopsis) subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways [1]. Specifically, 13C-isotopic labeling approaches showed that these WRF utilize poplar-derived aromatic compounds (e.g. 4-hydroxybenzoic acid (4-HBA)) as a carbon source. In silico genome analysis led us to hypothesize a complete catabolic pathway for 4-HBA and identify multiple homologous sequences for enzymes with putative oxidative decarboxylase, hydroxylase, and ring-opening dioxygenase activities, which are among the main biochemical reactions acting on aromatic compounds. Spatial and differential proteomic and metabolomic analyses supported the proposed catabolic pathways and showed alternative catabolic steps in T. versicolor that were not present in G. subvermispora. Based on the in silico, proteomics, and transcriptomics results, we down-selected enzymes for further in vitro characterization, and we have assigned a function to six fungal enzymes (including oxidative decarboxylases, hydroxylases, and ring-opening dioxygenases). Interestingly, even though we selected homologous enzyme pairs from both WRF with similar -omics trends, in a few cases only one of the studied fungi showed activity for the proposed substrate. Based on all the observations from this study [1], we hypothesized that 4-HBA preferentially undergoes oxidative decarboxylation to hydroquinone and subsequent hydroxylation to 1,2,4-benzenetriol in G. subvermispora before ring cleavage, whereas 4-HBA would preferentially undergo hydroxylation to protocatechuate and further oxidative decarboxylation to 1,2,4-benzenetriol in T. versicolor. Examining additional decarboxylases and hydroxylases as well as enzymes from other protein families that can perform the same or similar oxidative reactions, such as cytochromes P450 with aromatic hydroxylation activity, will also be key for elucidating enzyme preferences for specific substrates. Overall, this work forms the foundation of a new research area based on lignin catabolism by WRF, which could be further exploited to convert the undervalued biopolymer lignin into value-added compounds.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

DOE OSTI2023

HLA-Clus: HLA class I clustering based on 3D structure

In a previous paper, we classified populated HLA class I alleles into supertypes and subtypes based on the similarity of 3D landscape of peptide binding grooves, using newly defined structure distance metric and hierarchical clustering approach. Compared to other approaches, our method achieves higher correlation with peptide binding specificity, intra-cluster similarity (cohesion), and robustness. Here we introduce HLA-Clus, a Python package for clustering HLA Class I alleles using the method we developed recently and describe additional features including a new nearest neighbor clustering method that facilitates clustering based on user-defined criteria. The HLA-Clus pipeline includes three stages: First, HLA Class I structural models are coarse grained and transformed into clouds of labeled points. Second, similarities between alleles are determined using a newly defined structure distance metric that accounts for spatial and physicochemical similarities. Finally, alleles are clustered via hierarchical or nearest-neighbor approaches. We also interfaced HLA-Clus with the peptide:HLA affinity predictor MHCnuggets. By using the nearest neighbor clustering method to select optimal allele-specific deep learning models in MHCnuggets, the average accuracy of peptide binding prediction of rare alleles was improved. The HLA-Clus package offers a solution for characterizing the peptide binding specificities of a large number of HLA alleles. This method can be applied in HLA functional studies, such as the development of peptide affinity predictors, disease association studies, and HLA matching for grafting. HLA-Clus is freely available at our GitHub repository (https://github.com/yshen25/HLA-Clus).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Understanding the Structure and Dynamics of Complex Biomembrane Interactions by Neutron Scattering Techniques

The membrane is one of the key structural materials of biology at the cellular level. Composed predominantly of a bilayer of lipids with embedded and bound proteins, it defines the boundaries of the cell and many organelles essential to life and therefore is involved in almost all biological processes. Membrane-specific interactions, such as drug binding to a membrane receptor or the interactions of an antimicrobial compound with the lipid matrix of a pathogen membrane, are of interest across the scientific disciplines. Herein we present a review, aimed at nonexperts, of the major neutron scattering techniques used in membrane studies: small-angle neutron scattering, neutron membrane diffraction, neutron reflectometry, quasielastic neutron scattering, and neutron spin echo. Neutron scattering techniques are well suited to studying biological membranes. The nondestructive nature of cold neutrons means that samples can be measured for long periods without fear of beam damage from ultraviolet, electron, or X-ray radiation, and neutron beams are highly penetrating, thus offering flexibility in samples and sample environments. Most important is the strong difference in neutron scattering lengths between the two most abundant forms of hydrogen, protium and deuterium. Changing the relative amounts of protium/deuterium in a sample allows the production of a series of neutron scattering data sets, enabling the observation of differing components within complex membrane architectures. This approach can be as simple as using the naturally occurring neutron contrast between different biomolecules to study components in a complex by changing the solution H 2 O/D 2 O ratio or as complex as selectively labeling individual components with hydrogen isotopes. This review presents an overview of each experimental technique with the neutron instrument configuration, related sample preparation and sample environment, and data analysis, highlighted by a special emphasis on using prominent neutron contrast to understand structure and dynamics. This review gives researchers a practical introduction to the often enigmatic suite of neutron beamlines, thereby lowering the barrier to taking advantage of these large-facility techniques to achieve new understandings of membranes and their interactions with other molecules.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Backbone Dynamics of Bottlebrush Polymers Studied by Neutron Scattering

Bottlebrush polymers have a versatile architecture that is highly customizable due to the combination of a linear backbone and side chains. As a result of the chemical tethering of the side chains, both parts cannot be easily be separated. Huge effort is seen regarding the dynamical behavior of the side chains or the entire bottlebrush polymer, whereas few studies are available considering the backbone. Here, isotopic labeling in combination with quasi-elastic neutron scattering was used to compare the dynamical behavior of the bottlebrush’s backbone with the side chain dynamics. Keeping the side chains deuterated, (h-PNB)- g -(d-PPO), leads to the scattering signal dominated by backbone dynamics, while the fully protonated sample, (h-PNB)- g -(h-PPO), gives side chain dynamics. Both results reveal slower dynamics associated with the backbone with less heterogeneity, as seen for the side chains. Additionally, a plasticizer effect for the backbone dynamics is confirmed by extracting the glass transition temperature and comparing it with pure linear PNB.

neutron scattering↗

DOE OSTI2025

DNA-PAINT Imaging with Hydrogel Imprinting and Clearing

Hydrogel-embedding is a versatile technique in fluorescence microscopy, offering stabilization, optical clearing, and the physical expansion of biological specimens. DNA-PAINT is a super-resolution microscopy approach based on the diffusion and transient binding of fluorescently labeled oligos, but its feasibility in hydrogels has not yet been explored. In this study, we demonstrate that polyacrylamide hydrogels support sufficient diffusion for effective DNA-PAINT imaging. Using acrydite-anchored oligonucleotides imprinted from patterned DNA origami nanostructures and microtubule filaments in fixed cells, we find that hydrogel embedding preserves docking strand positioning at the nanoscale. Sample clearing via protease treatment had minor structural effects on the microtubule structure and enhanced diffusion and accessibility to hydrogel-imprinted docking strands. Our work demonstrates promising potential for diffusion and binding-based fluorescence imaging applications in hydrogel-embedded samples.

DNA origami↗

NASA NTRS2001

Mechanical behavior in living cells consistent with the tensegrity model

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

NASA Discipline Cell Biology↗

NASA NTRS2002

Clinostat rotation induces apoptosis in luteal cells of the pregnant rat

Recent studies have shown that microgravity induces changes at the cellular level, including apoptosis. However, it is unknown whether microgravity affects luteal cell function. This study was performed to assess whether microgravity conditions generated by clinostat rotation induce apoptosis and affect steroidogenesis by luteal cells. Luteal cells isolated from the corpora lutea of Day 8 pregnant rats were placed in equal numbers in slide flasks (chamber slides). One slide flask was placed in the clinostat and the other served as a stationary control. At 48 h in the clinostat, whereas the levels of progesterone and total cellular protein decreased, the number of shrunken cells increased. To determine whether apoptosis occurred in shrunken cells, Comet and TUNEL assays were performed. At 48 h, the percentage of apoptotic cells in the clinostat increased compared with that in the control. To investigate how the microgravity conditions induce apoptosis, the active mitochondria in luteal cells were detected with JC-1 dye. Cells in the control consisted of many active mitochondria, which were evenly distributed throughout the cell. In contrast, cells in the clinostat displayed fewer active mitochondria, which were distributed either to the outer edge of the cell or around the nucleus. These results suggest that mitochondrial dysfunction induced by clinostat rotation could lead to apoptosis in luteal cells and suppression of progesterone production.

NASA Discipline Cell Biology↗

DOE OSTI2026

Precursor-Dependent Routing of Aromatic Amino Acids Determines Lignin Structure in Grasses by Sensitivity-Enhanced Solid-State NMR

Lignin biosynthesis in grasses exhibits unique metabolic flexibility, yet the precursor-specific routing of carbon into lignin polymers remains poorly resolved in planta. Here, we combine 13 C-isotope labeling with solid-state NMR under sensitivity-enhancement by dynamic nuclear polarization (DNP), to directly track phenylalanine- and tyrosine-derived carbon incorporation into the lignin polymer in Brachypodium distachyon. Precursor-specific 13 C labeling reveals that phenylalanine is the dominant contributor to canonical guaiacyl and syringyl lignins, whereas tyrosine preferentially enriches hydroxyphenyl lignin and hydroxycinnamates, including ferulates characteristic of grass cell walls. Two-dimensional 13 C− 13 C correlation NMR resolves distinct lignin moieties arising from each precursor. Disruption of pcoumarate 3-hydroxylase (C3H) selectively impairs phenylalanine-derived lignification, while tyrosine-derived lignin remains comparatively unchanged, maintaining polymer assembly through alternative metabolic routes. These findings show precursordependent control of lignin composition and reveal tyrosine-mediated lignification as a compensatory pathway in grasses. This work also establishes precursor-resolved solid-state NMR and DNP as a powerful framework for dissecting lignin biosynthesis and metabolic plasticity in plant cell walls.

Biopolymers↗