Dynamic Regulation of Histone Chaperone Nucleoplasmin

Small-angle X-ray scattering (SAXS) analysis of nucleoplasmin (Npm) Core+A2 truncation (1-145) bound to five H2A/H2B dimers. (Top) SAXs envelope of the pentameric complex (pink) with the best nuclear magnetic resonance (NMR)–restrained SAXS hybrid model inside. (Bottom) SAXS curve of the complex (purple dots). Simulated SAXS curve (black line) from the best-scoring structural model. [From Warren, C., et al. “Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.” Nat. Commun. 8, 2215 (2017). DOI:10.1038/s41467-017-02308-3. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/​).]
Histones are eukaryotic cell nuclei proteins that package and order DNA into structural units called nucleosomes. Chromatin is the complex of DNA and proteins comprising the genome’s physiological form. As chromatin’s chief protein components, histones act as spools around which DNA winds, playing a role in gene regulation. A chaperone protein assists in the folding and unfolding of macromolecules, such as in the assembly of nucleosomes from folded histones and DNA. Nucleoplasmin (Npm) is a highly conserved embryonic histone chaperone, responsible for the maternal storage and zygotic release of histones H2A and H2B. Npm contains a pentameric N-terminal Core domain and an intrinsically disordered C-terminal Tail domain. Although intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning have remained unclear.

This study, using the Xenopus laevis Npm Tail domain, unveils the architecture of the Npm histone complex and a mechanism of histone chaperone regulation. It demonstrates that intramolecular regulation of the histone chaperone Npm controls histone binding and release—a key process in the earliest stages of embryonic development. Structural analyses enabled model constructions of both the Npm Tail domain and the pentameric complex, revealing that the Tail domain controls the binding of histones through specific, electrostatic interactions. Functional analyses demonstrated that these competitive interactions negatively regulate Npm histone chaperone activity in vitro. Data from these studies establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.

Warren, C., et al. “Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.” Nat. Commun. 8, 2215 (2017). DOI:10.1038/s41467-017-02308-3.

Instruments and Facilities Used: Bruker 600 nuclear magnetic resonance (NMR) and Inova 600 NMR instruments in the Albert Einstein College of Medicine (AECM) Einstein Structural NMR Resource; and bio–small-angle X-ray scattering (bio-SAXS) beamline 4-2, SLAC National Accelerator Laboratory’s Stanford Synchrotron Radiation Lightsource (SSRL).

Funding Acknowledgements: Supported by The American Cancer Society (ACS)Robbie Sue Mudd Kidney Cancer Research Scholar Grant (124891-RSG-13-396-01-DMC) and National Institutes of Health (NIH) grant R01GM108646 (both to D.S.) and training grants T32GM007491 and F31GM116536 (to C.W). J.M.K. supported by Einstein Medical Scientist Training Program Grant (T32 GM007288). Bruker 600 nuclear magnetic resonance (NMR) instrument purchased using funds from NIH award 1S10OD016305 and supported by Albert Einstein College of Medicine (AECM). Inova 600 NMR instrument in the Einstein Structural NMR Resource purchased using funds from NIH award 1S10RR017998 and National Science Foundation (NSF) award DBI0331934 and supported by the AECM. Use of Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory (SLAC), supported by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under Contract No. DE-AC02-76SF00515. SSRL Structural Molecular Biology Program supported by the Office of Biological and Environmental Research (OBER), DOE Office of Science, and by National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; including P41GM103393).

X-Ray Footprinting Solves Mystery of Metal-Breathing Protein

Participants in protein-wiring study, LBNL
Participants in a protein-wiring study at Lawrence Berkeley National Laboratory included (from left): Jose Cornejo, Corie Ralston, Caroline Ajo-Franklin, Sayan Gupta, and Behzad Rad. Not pictured: Tatsuya Fukushima, Christopher Petzold, Leanne Chan, and Rena Mizrahi. [Courtesy Paul Mueller, Lawrence Berkeley National Laboratory]
Caroline Ajo-Franklin, a staff scientist in the Biological Nanostructures Facility at Lawrence Berkeley National Laboratory’s (LBNL) Molecular Foundry (one of the Nanoscale Science Research Centers supported by DOE’s Office of Basic Energy Sciences), teamed up with Corie Ralston to use X-ray mass spectrometry footprinting at LBNL’s Advanced Light Source. Ralston, who works in the Molecular Biophysics and Integrated Bioimaging (MBIB) Division, uses the X-ray mass spectrometry footprinting technique to precisely probe proteins and their surroundings at the Advanced Light Source. Ajo-Franklin and Ralston saw that they could use footprinting to answer a long-standing question in microbiology: how do bacterial proteins interact directly with minerals to transfer electrons and allow the microbe to live?

“Understanding what these interactions between proteins and materials look like can help us design them better,” Ajo-Franklin said, “and give us insight on how to connect living cells with devices.”

Surprisingly, “the biggest finding … was that our proteins bind relatively weakly,” Ajo-Franklin noted. “Most proteins that interface with materials bind really tightly,” changing shape as they form the connection. This particular protein does not appear to change shape at all and only interacts with the mineral in a small area, requiring about five times less binding energy, by comparison, than typical proteins that form biominerals. This finding makes a lot of sense, Ajo-Franklin, because this protein’s job “is to transfer electrons to the mineral, so it doesn’t have to be in contact for very long.”

Beamline contact:
Corie Ralston
Advanced Light Source

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What’s on Your Skin? Archaea, That’s What

Hoi-Ying Holman, Advanced Light Source, LBNL
As director of the Berkeley Synchrotron Infrared Structural Biology Imaging Program at the Advanced Light Source, Hoi-Ying Holman focuses on developing and providing research communities with new synchrotron infrared technologies for deciphering the relationship between genome and functional processes and identifying the connection between the genome and natural environments. [Courtesy Marilyn Chung, Lawrence Berkeley National Laboratory]
To characterize microbes on human skin, researchers from Austria, including Christine Moissl-Eichinger, collaborated with Hoi-Ying Holman and other scientists at the Berkeley Synchrotron Infrared Structural Biology Imaging Program, a BER-supported infrared beamline at the Advanced Light Source at Lawrence Berkeley National Laboratory. The research stemmed from a joint project between the National Aeronautics and Space Administration and the European Space Agency.

“We were checking spacecraft and their clean rooms for the presence of archaea, as they are suspected to be possible critical contaminants during space exploration,” Moissl-Eichinger said. “Certain methane-producing archaea, the so-called methanogens, could possibly survive on Mars. We did not find many signatures from methanogens, but we found loads of Thaumarchaeota, a very different type of archaea that survives with oxygen.”

This finding led to the discovery that these archaea are present on people’s skin. The infrared beamline was used to rapidly and precisely characterize samples from humans and determine the levels and types of microbes present, based on the chemical specificity of infrared spectroscopy. This analysis could then be linked back to the genomic data collected by the Austrian team. The detected archaea are probably involved in nitrogen turnover on skin and are capable of lowering skin pH, supporting the suppression of pathogens. The researchers found that because of changes in skin moisture, these microbes are most abundant in subjects younger than 12 and older than 60.

Beamline contact:
Hoi-Ying Holman
Berkeley Synchrotron Infrared Structural Biology Imaging Program
Advanced Light Source

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“Zip-code” Mechanism for Hormone Signaling Revealed

Crystal structure of multikinase inhibitor sKLB bound to fibroblast growth factor (FGF) FGF21CT reveals two distinct binding sites. Structure of extracellular domain of β-Klotho “longevity” protein (green ribbons) bound to FGF21 hormone (salmon ball-and-stick) unveils critical molecular interactions required for hormone binding and cell activation. Yellow sticks denote nitrogen (N)-linked glycans. Grey dashed lines denote regions that do not exhibit significant electron density. About 30 angsroms (Å) apart, the FGF binding sites on the β-Klotho D1 and D2 domains are located on the opposite side of the molecule from the flexible linker that connects the two glycosidase domains and may contribute to the interdomain dynamic properties that enable complex formation with ligands and FGF receptors (FGFRs). [Reprinted by permission from Springer Nature: Lee, S., et al. “Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signaling.” Nature 553, 501–505 (2018). DOI:10.1038/nature25010. Copyright 2018]
Named after the Greek goddess who spun the thread of life, Klotho proteins play an important role in the regulation of longevity and metabolism. Alpha-Klotho is a membrane-spanning protein expressed predominantly in the kidney, as well as in the brain. Mice lacking α-klotho exhibit a range of signs associated with aging and have elevated blood phosphate levels. Like α-klotho, β-klotho functions as a co-receptor for endocrine fibroblast growth factors (FGFs). FGF21 is secreted from the liver following fasting, acting in fat cells and the brain to induce metabolic adaptation to fasting and responses to stress. Although FGF receptors (FGFRs) are expressed in a wide range of tissues, expression of the β-klotho “longevity” protein in the liver, fat, and brain restricts the target organs of these endocrine FGFs.

In a recent Yale Medical School–led study, researchers revealed the three-dimensional (3D), high-resolution structure of β-Klotho, illuminating its intricate mechanism and potential for antiaging therapeutics, as well as for treating a wide range of medical conditions. X-ray crystallography data revealed critical molecular interactions required for hormone binding and cell activation. The specific “zip-code”–like interactions of β-klotho receptors appear to regulate critical metabolic processes in the liver, kidneys, and brain, among other organs. Analysis yielded several insights, including that β-Klotho is the primary receptor that binds to FGF21, a key hormone that stimulates insulin sensitivity and glucose metabolism, causing weight loss. The researchers believe this new understanding can guide the development of therapies by improving the biological activity of FGF21. Also found were a new variant of FGF21 that has 10 times higher potency and cellular activity and the evidence of how a structurally related enzyme (glycosidase) that breaks down sugars evolved into a receptor for a hormone that lowers blood sugar. Researchers believe the untangled β-Klotho structure presents a platform for exploring and developing agents that either enhance or block the pathway, enabling therapies for conditions such as liver cancer and bone diseases.

Lee, S., et al. “Structures of β-klotho reveal a ‘zip code’-like mechanism for endocrine FGF signaling.” Nature 553, 501–505 (2018). DOI:10.1038/nature25010.

Instruments and Facilities Used: X-ray data were collected from β-klotho–receptor crystals at Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory, beamline 14-1 as part of the program to aid National Synchrotron Light Source (NSLS) users prior to NSLS-II operations.

Funding Acknowledgements: The National Synchrotron Lightsource (NSLS) Stanford Synchrotron Radiation Lightsource (SSRL) supported by P41GM111244, P41GM103393, DE-SC0012704, and DE-AC02-76SF00515. NE-CAT (P41 GM103403) and APS (DE-AC02-06CH11357). Research also supported by National Institutes of Health (NIH) grant 1S10OD018007 and NIH Award S10RR026992-0110. J.St. financial support: INSTRUCT (European Strategy Forum on Research Infrastructures [ESFRI], participate with or without funding [FWO]).

Structure of a Flavoenzyme Assembly Intermediate

(a) The structure of the FrdA-SdhE flavoenzyme assembly intermediate: flavoprotein subunit FrdA (cyan), assembly factor SdhE (green), flavin adenine dinucleotide FAD (orange sticks), and malonate (yellow sticks). The boxed region highlights the covalent interaction between the FAD and the enzyme. (b) Overlay of the flavin-binding domains of the FrdA subunit from the FrdA-SdhE intermediate (cyan) and the FrdA subunit from the mature assembled FrdABCD complex (gray). A rotation of 10.8° is observed in the capping domain of the assembly intermediate when compared to assembled FrdABCD. [From Sharma, P., et al. “Crystal structure of an assembly intermediate of respiratory Complex II,” Nat. Commun. 9, 274 (2018). DOI:10.1038/s41467-017-02713-8. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/​.)]
Enzymes frequently depend on an electron transport cofactor for executing catalytic functions such as reduction-oxidation (redox) reactions. For flavoenzymes, the cofactor is flavin adenine dinucleotide (FAD), whose binding type with the enzyme impacts the redox potential and thus reaction chemistry, such as for metabolism and detoxification. Researchers in this study discovered that the structure of an assembled flavoenzyme intermediate reveals the mechanism of covalent flavin binding in respiration. Assembly factors include SdhAF2 in humans, SdhE in Escherichia coli, and Sdh5 in yeast. Other revelations include that mitochondrial flavoenzymes drive both noncovalent and covalent redox reactions and that the assembly factor (SdhE, a small protein of ~90 to 140 amino acids, conserved in all kingdoms) in the structure of the SdhE:FrdA complex with covalent FAD stabilizes a conformation of the flavoprotein subunit FrdA that favors succinate oxidation.

Researchers fixed the E. coli FrdA-SdhE intermediate via site-specific crosslinking, resolving the structure to 2.6 angstroms (Å). This study identified that SdhE stabilizes an FrdA conformation that likely enables the mechanism of autocatalytic covalent flavinylation. FrdA’s FAD-binding domain and capping domain both interact with SdhE, but structural data revealed a 10.8° difference in their angles. The investigators believe that domain rotation affects flavinylation, showing that enzymes are tuned to catalyze reactions in different ways and that conformational diversity can directly relate to catalytic mechanism diversity.

Sharma, P., et al. “Crystal structure of an assembly intermediate of respiratory Complex II.” Nat. Commun. 9, 274 (2018). DOI:10.1038/s41467-017-02713-8.

Instruments and Facilities Used: Small angle X-ray scattering (SAXS) and diffraction and mass spectrometry analysis using beamline 9-2 at Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC National Accelerator Laboratory (SLAC).

Funding Acknowledgements: Supported by Department of Veterans Affairs (DVA; BX001077 to G.C.) and National Institutes of Health (NIH; GM061606 to G.C. and T.M.I.). G.C. (recipient of a Senior Research Career Scientist award, #IK6B004215 from DVA). Vanderbilt University crystallization facility support: S10 RR026915. Use of Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory (SLAC), supported by Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under Contract No. DE-AC02-76SF00515. SSRL Structural Molecular Biology Program supported by Office of Biological and Environmental Research (OBER), DOE Office of Science, and by the NIH National Institute of General Medical Sciences (NIGMS; including P41GM103393).

Biological Small Angle Scattering: Techniques, Strategies and Tips

“Biological Small Angle Scattering: Techniques, Strategies and Tips” edited by B Chaudhuri, IG Muñoz, S Qian, VS Urban was published by Springer (ISBN 978-981-10-6038-0) DOI: 10.1007/978-981-10-6038-0

Bio-SANS scientists S. Qian, V. Urban along with B. Chaudhuri (an early Bio-SANS user) and I. Munoz compiled the first introductory book on biological solution small angle scattering (SAS). Biological SAS has seen tremendous growth over the past decade because it is especially useful for studying proteins which in turn may have big implications for renewable energy, medical care and drug effectiveness. The chapters are written by international experts in solution SAS methodologies, many from US user facilities (SSRL, NCNR, LBNL). The carefully selected topics include techniques for improving data quality and analysis, as well as different scientific applications of SAS. The book includes the principles and theoretical background of various SAS techniques and practical aspects that range from sample preparation to data publication. It is a handbook for any researcher using X-ray/neutron small-angle scattering in biology.

Grand Challenges for Biological and Environmental Research: Progress and Future Vision

The Biological and Environmental Research (BER) program within the U.S. Department of Energy (DOE) Office of Science supports research focusing on the interconnections between energy production and the living environment. This fundamental research, conducted at universities, DOE national laboratories, and research institutions across the country, explores organisms and ecosystems that can influence the U.S. energy system and advances understanding of the relationships between energy and environment from local to global scales.

A report from the Biological and Environmental Research Advisory Committee

BER regularly solicits input from the scientific community to help guide its programs. The Biological and Environmental Research Advisory Committee (BERAC) is chartered under the Federal Advisory Committee Act to advise BER on its research portfolio and user facilities. To facilitate a synthesis of community input, the director of DOE’s Office of Science charged BERAC in March 2016 to review research progress and establish and deliver a revised long-term vision for BER by fall 2017. Questions considered during this process included:

  • To what extent has BER successfully met the challenges outlined in the 2010 report, Grand Challenges for Biological and Environmental Research: A Long-Term Vision?
  • What are the greatest scientific challenges that DOE faces in the long term (20-year horizon), and for which of these should BER take primary responsibility?
  • How should DOE position BER to address these challenges?
  • What new tools should be developed to integrate and analyze data from different disciplines?
  • What unique opportunities exist to partner with, or leverage assets from, other programs within the DOE Office of Science?
  • What scientific and technical advances are needed to train the future workforce in integrative science, including complex systems science?

Through a series of BERAC meetings, white papers, and a research community workshop, BERAC addressed these questions, identifying future grand challenges in five areas: biological systems, Earth and environmental systems, microbial to Earth system pathways, energy sustainability, and data analytics and computing. Providing critical support for these challenges are BER user facilities, research infrastructure, and emerging technologies. This report represents a synthesis of these grand challenges and the supporting facilities and technologies.

Publication date: November 2017

Suggested citation for this report: BERAC. 2017. Grand Challenges for Biological and Environmental Research: Progress and Future Vision; A Report from the Biological and Environmental Research Advisory Committee, DOE/SC–0190, BERAC Subcommittee on Grand Research Challenges for Biological and Environmental Research (science.energy.gov/~/media/ber/berac/pdf/Reports/BERAC-2017-Grand-Challenges-Report.pdf).

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A Regional Model for Uranium Redox and Mobility

Anoxic organic-enriched sediments strongly accumulate uranium as U(IV), sulfide, and other reduced species. Seasonal reduction-oxidation (redox) cycling triggered by changing water tables can intermittently mobilize these species. [Courtesy Vincent Noël and John Bargar, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory]
Uranium (U) contamination stubbornly persists as a challenging and costly water quality concern at former uranium ore processing sites across the Upper Colorado River Basin (UCRB). Plumes at these sites are not self-attenuating via natural flushing by groundwater as originally expected. Recent studies at the Rifle, Colo., legacy site suggest that organic-enriched anoxic sediments create conditions that promote reduction of U(VI) to relatively immobile U(IV), causing it to accumulate locally under persistently saturated and anoxic conditions. However, incursion of oxidants into reduced sediments could transform contaminants, allowing these sediments to act as secondary sources of uranium. Oxidant incursions take place during periods of changing water tables, which occur in UCRB throughout the year. If these sediments were regionally common in the UCRB and exposed to varying reduction-oxidation (redox) conditions, then they could contribute to maintaining the longevity of regional uranium plumes.

To investigate these issues, researchers examined the occurrence and distribution of reduced and oxidized iron (Fe), sulfur (S), and U species in sediment cores spanning dry and oxic to wet and reduced conditions at three different UCRB sites. Detailed molecular characterization involved chemical extractions, X-ray absorption spectroscopy (XAS), Mössbauer spectroscopy, and X-ray microspectroscopy. This work demonstrates that anoxic organic-enriched sediments occur at all sites, strongly accumulate sulfides and U, and are exposed to strong seasonal redox cycles. Uranium was found to be present as U(IV) complexed to sediment-associated organic carbon and possibly to mineral surfaces. This finding is significant because complexed U(IV) is relatively susceptible to oxidative mobilization. Sediment particle size, organic carbon content, and pore saturation control redox conditions in sediments and thus strongly influence Fe, S, and U biogeochemistry. These findings help to illuminate the mechanistic linkages between hydrology, sediment texture, and biogeochemistry. They further provide enhanced contextual and conceptual underpinnings to support reactive transport modeling of uranium, other contaminants, and nutrients in redox-variable floodplains—a subject of importance to BER research missions. Cyclic redox variability has major implications for mobility of carbon (C), nitrogen (N), and metal contaminants in groundwater and surface waters. Redox-variable, organic-enriched sediments mediate the mobility of C, N, Fe, S, U, and metal contaminants regionally in the UCRB. Organic-enriched sediments were established to regionally mediate groundwater quality within the UCRB.

Noël, V., et al. “Understanding controls on redox processes in floodplain sediments of the Upper Colorado River Basin.” Sci. Total Environ. 603–604, 663–675 (2017). DOI:10.1016/j.scitotenv.2017.01.109.

Noël, V., et al. “Redox constraints over U(IV) mobility in the floodplains of Upper Colorado River Basin.” Environ. Sci. Technol. 51(19), 10954–10964 (2017). DOI:10.1021/acs.est.7b02203.

Instruments and Facilities Used: X-ray absorption spectroscopy and X-ray microprobe mapping at Stanford Synchrotron Radiation Lightsource (SSRL), SLAC National Accelerator Laboratory (SLAC), and Mössbauer spectroscopy at Environmental Molecular Sciences Laboratory (EMSL), Pacific Northwest National Laboratory (PNNL).

Funding Acknowledgements:

For “Understanding controls on redox processes in floodplain sediments of the Upper Colorado River Basin.” Sci. Total Environ. DOI: 10.1016/j.scitotenv.2017.01.109: Supported by the Office of Biological and Environmental Research (OBER) Climate and Environmental Sciences Division, U.S. Department of Energy (DOE) Office of Science, through the SLAC National Accelerator Laboratory (SLAC) Science Focus Area (SFA) program and by DOE Office of Science’s Office of Basic Energy Sciences-(OBES) through its support for Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC. SSRL and SLAC are supported by OBES, DOE Office of Science, under Contract No. DE-AC02-76SF00515 and OBER, and by the National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; including P41GM103393). Material partially based on work supported through Lawrence Berkeley National Laboratory’s (LBNL) Genomes-to-Watershed Scientific Focus Area (SFA). The DOE Office of Science’s, OBER-funded work under contract DE-AC02-05CH11231 (LBNL; operated by the University of California).

For “Redox constraints over U(IV) mobility in the floodplains of Upper Colorado River Basin.” Environ. Sci. Technol. DOI: 10.1021/acs.est.7b02203: X-ray fluorescence spectrometry with a XEPOS (Spectro X Lab) x-ray fluorescence spectrometer. x-ray synchrotron powder x-ray diffraction (SR-XRD) performed on sediments as part of this study. Porewater and groundwater samples analyzed for their element composition using high-resolution inductively coupled plasma mass spectroscopy (HR-ICP-MS), X-ray Absorption Spectroscopy (XAS). U LII-edge x-ray absorption near-edge structure (XANES) spectroscopy used to determine uranium (U) oxidation states. Research supported by the Office of Biological and Environmental Research (OBER) Climate and Environmental Sciences Division, U.S. Department of Energy (DOE) Office of Science, through the SLAC National Accelerator Laboratory (SLAC) Science Focus Area (SFA) program and by the Office of Basic Energy Sciences (OBES), DOE Office of Science, through its support for Stanford Synchrotron Radiation Lightsource (SSRL) at SLAC. SSRL and SLAC are supported by OBES, DOE Office of Science, under Contract No. DE-AC02-76SF00515; OBER; and National Institutes of Health’s (NIH) National Institute of General Medical Sciences (NIGMS; including P41GM103393). Coring and field site access provided by DOE’s Legacy Management. Field work at the Rifle site partially supported by the Lawrence Berkeley National Laboratory’s (LBNL) Genomes-to-Watershed SFA. The DOE Office of Science OBERfunded work is under contract DE-AC02-05CH11231 (LBNL, operated by the University of California). The Canadian Light Source (CLS) is supported by Natural Sciences and Engineering Research Council of Canada, National Research Council Canada, Canadian Institutes of Health Research, Province of Saskatchewan, Western Economic Diversification Canada, and University of Saskatchewan.

Cyanobacterial Studies Examine Cellular Structure During Nitrogen Starvation

Researchers from Washington University in St. Louis and Oak Ridge National Laboratory (ORNL) are using neutrons to study what happens when cyanobacteria cell samples (pictured) are starved for nitrogen. They are especially interested in how this process affects phycobilisomes, large antenna protein complexes in the cells that harvest light for photosynthesis. A better understanding of this natural phenomenon could lead to improvements in artificial resources like solar panels. [Courtesy ORNL]
Using nondestructive neutron scattering techniques, scientists are examining how single-celled organisms called cyanobacteria produce oxygen and obtain energy through photosynthesis. Collaborators are conducting a series of experiments to study the behavior of phycobilisomes—large antenna protein complexes in cyanobacteria cells. Phycobilisomes harvest light to initiate photosynthesis, and a better understanding of this process could help researchers design more efficient solar panels and other artificial structures that mimic natural systems. Neutrons can analyze these delicate structures without damaging or killing the cyanobacteria and with more spatial accuracy than other techniques like microscopy. Bio-SANS allows observing what’s happening at the nanoscale level in real time in a living cell.

Phycobilisomes attach to cellular membranes where the light-dependent reactions of photosynthesis take place. Changing the antenna complexes of the phycobilisomes can have dramatic and far-reaching consequences in cyanobacteria. Artificially modifying phycobilisomes by deleting certain genes in the cells caused structural defects in the cellular membranes and other cell physiology, allowing scientists to observe the resulting structural changes.

Starving the cyanobacteria for nitrogen naturally modifies the antenna complexes, causing the antenna to decrease in size and leading to significant cellular membrane modifications, because the cells break down the phycobilisomes and use them as an alternative nitrogen source to survive. By determining the extent of these changes, the team hopes to better understand the structure-function relationship between cellular organization and natural modification. These processes can be immediately reversed by restoring nitrogen to the cells. The researchers plan to compare these results to those recorded from their genetic studies to explore the differences between artificial and natural modifications and their effects on the intracellular makeup of cyanobacteria.

Instruments and Facilities Used: Photosynthetic Antenna Research Center (PARC), a DOE BES-funded Energy Frontier Research Center based at Washington University at St Louis. Small angle neutron scattering (SANS) was performed at the DOE-BER supported Bio-SANS instrument, beamline CG‑3, at Oak Ridge National Laboratory’s High Flux Isotope Reactor.

Technologies for Characterizing Molecular and Cellular Systems Relevant to Bioenergy and Environment

Report from the September 2016 Workshop

The Biological Systems Science Division (BSSD) within the U.S. Department of Energy’s (DOE) Office of Biological and Environmental Research (BER) funds basic research on plants and microbes relevant to several DOE bioenergy and environmental mission areas.BSSD research seeks to understand the fundamental genome-encoded properties of plants and microbes that can be harnessed or redesigned for beneficial purposes.

Current emphases are leading to the discovery, development, and understanding of numerous plant and microbial species with traits suitable for the production of fuels and chemical products from renewable biomass that could be grown synergistically with food or animal feed crops while not competing with other societal needs. Additionally, BSSD further supports research leading to an understanding of the complex and essential interactions among plants, microbial communities, and the environment to find new ways to sustainably produce biomass for a range of bioenergy and bioproduct applications. This research also is relevant for incorporation into larger-scale environmental models such as those developed through the research supported by BER’s Earth and Environmental Systems Sciences programs.

To engage the relevant scientific communities in discussions of these research areas, BER convened the Technologies for Characterizing Molecular and Cellular Systems Relevant to Bioenergy and Environment workshop on September 21–23, 2016. Seeking to enable more comprehensive systems biology–based approaches, which typically require measurements of many samples, workshop participants highlighted the need for the development of highly sensitive methods to provide accurate measurements from small sample volumes and that are operable in high-throughput or highly parallel modes.

Publication date: September 2017

Suggested citation for this report: U.S. DOE. 2017. Technologies for Characterizing Molecular and Cellular Systems Relevant to Bioenergy and Environment, DOE/SC-0189, U.S. Department of Energy Office of Science. (https://genomicscience.energy.gov/technologies/).

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Research Themes

These fundamental research efforts require new and innovative methods and technologies to elucidate the foundational principles that drive biological systems of interest to DOE’s energy and environmental missions. Characterizing biological systems involves analytical approaches that illuminate cellular components and their form, structure, size, function, spatial location, dynamics, and interactions with the environment. Workshop discussions identified new technologies and combinations of existing capabilities to address the challenges associated with characterizing molecular and cellular systems relevant to bioenergy and environmental research. Participants included technology developers and biology researchers with expertise in cellular ultrastructure and physiology, bioenergy and bioproducts, and environmental microbiology. Attendees developed a series of research and technology development needs across six thematic areas spanning the range of BSSD-supported research. The challenges of studying these systems are many and broad in scope, covering time scales from femtoseconds to weeks and length scales from Angstroms to centimeters. This report addresses this very broad measurement range—from cells and their metabolism and mineralogy (Angstroms to micrometers), to rhizosphere ecosystem processes and community biochemical activity (millimeters to a meter). In this context, the range from micrometers to a meter is referred to as “mesoscale.” Despite the breadth of the challenges, participants identified key needed technologies and improvements in current techniques that could advance BER science. These six major research themes are discussed below.

  • Cell Wall Composition and Degradation. The benefits from gaining a molecular-level understanding of plant cell wall composition and degradation were discussed in the context of using plants in the production of biofuels and bioproducts. As a renewable resource for biofuels and biomaterials, lignocellulosic biomass can partially replace the use of diminishing petroleum-based fuels and products and help meet increasing consumer demand for green chemicals. However, the varying structure and chemical composition of the cell walls of different plants and tissue types may hinder industrial-scale processes for converting biomass to bioproducts. Needed to address this challenge are better atomic- and molecular-level understandings of the structure and dynamics of naturally occurring cell wall processes, as well as the processes involved in the production of biofuels and other chemicals. Also needed are new characterization techniques with nanometer-scale resolution that require minimal sample preparation and keep the sample in close-to-natural conditions. Several technologies and techniques identified will aid the understanding of plant cell wall properties at the anatomical, cellular, molecular, and genetic levels. Other new approaches suggested will provide as-yet-undiscovered molecular details about structural and temporal rearrangement of cell wall components during biomass deconstruction prior to conversion to biofuels and bioproducts.
  • Rhizosphere Community Interactions. A better understanding of ecosystems is yielding deeper insights into plant-microbe-mineral interactions important for bioenergy production. Knowing the complex interdependencies of these three systems is critical to understanding and developing sustainable biofuel production practices. Root system architecture has a dramatic bearing on plant viability and crop productivity in given soil conditions. Namely, the rhizosphere, the area immediately surrounding plant roots, is a nexus of biological activity and the foundational ecosystem for any plant-microbe system. Thus, studies are needed of all essential communal elements necessary for plant growth and yield across a range of geographic regions. Understanding these ecosystems can enable the design of optimally mutualistic plant-microbe interactions to improve biofuel crop sustainability. For these studies, the development of penetrating imaging tools is needed to study entire, complex soil environments and root system architectures.
  • Biogeochemical Cycling. Environmental system function is intimately tied to the biogeochemical cycling of the major elements, particularly their reduction-oxidation (redox) transformations. Spatial and temporal imaging and measurements of biogeochemical systems are necessary for a mechanistic understanding of how different biogeochemical systems function. There also is a need for development of, and improvements to, technologies and approaches that will enable researchers (1) to understand and predict the dynamic interplay between environmental biotic and abiotic factors that often are opaque to imaging tools, from the molecular to the mesoscale, and (2) to use this new understanding to predict larger-scale phenomena. A combined and holistic use of a variety of dynamic imaging and characterization probes, coupled with multiomic and modeling approaches, is necessary to span spatial and temporal scales in biogeochemical systems to better understand their role in key environmental processes.
  • Metabolic Pathways in Plants, Microbes, and Fungi. Plants and microbes exchange metabolites in a community economy that ultimately determines the rates at which nutrients and water are extracted from soil and soil carbon is cycled (i.e., the biogeochemistry). A deeper understanding of the mechanisms by which organisms interact with each other in the environment, and the metabolic pathways and specific molecules involved in these interactions, will enable the modification of these pathways to improve nutrient-use efficiency and soil-carbon performance. New tools are required for predicting and measuring metabolites from organisms key to BER bioenergy and environmental missions. Combined with new higher-resolution approaches, these technologies will need capabilities for determining spatiotemporal localization and mechanisms responsible for metabolite synthesis, transport, degradation, and perception. The ultimate goal is a “balanced record” of metabolite economy among plant-microbe-fungi interactions and the environment that fully accounts for all carbon and nutrient cycling in the system.
  • Biosystems Design. Synthetic biology provides a valuable approach to probe, study, and engineer new functions into biological systems through the introduction or modification of metabolic pathways, specifically generating biologically derived chemicals, fuels, and materials to ensure environmental sustainability. Challenges include (1) applying synthetic biology to intractable eukaryotic and multicellular organisms, (2) engineering communities of microorganisms and microbe-plant interfaces, (3) exploring genotype-phenotype landscapes resulting from genome engineering, (4) isolating engineered organisms with desired functions, and (5) safeguarding engineered biosystems. Efficient tools for the precise manipulation of genomes in diverse target organisms will need to be combined with improved computational modeling methods to support predictive biology. These coupled approaches will require assistance from new methods for rapidly assaying function and fitness. They also must be applicable to technologies for controlling the containment of engineered systems and the products of engineered pathways.
  • Cellular Ultrastructure and Physiology. BER research examines a range of plant and microbial cell structures and organization, from the atomic level to complex molecular machines, cellular compartments, scaffolds, and whole cells. Workshop participants identified several measurement challenges, including how to detect and visualize cellular dynamic processes such as metabolic cycles, signaling and trafficking in plants, and interactions among microbial and fungal communities. They described needs for improved (1) structural imaging at the atomic and molecular level, (2) methods for illuminating whole organisms to understand the internal organization of cells, and (3) imaging chemical events that underlie biology. These needs include methods to determine the locations and dynamic parameters of enzyme reactions within cells, as well as the flow of chemicals and macromolecules within and between cells. The structural and dynamical insights from such studies will inform and enable more accurate modeling of biogeochemical cycling and metabolic pathways important in rhizospheric communities and biofuel or bioproduct processes.

Overarching Challenges and Opportunities

Fig. 9.2. Spatial and Temporal Resolutions of Imaging Technologies. Summary of imaging and other selected measurement technologies discussed at the workshop. Mature technologies that will benefit from further development include X-ray and neutron crystallography and scattering, scanning probe microscopies, X-ray tomography, synchrotron spectroscopy, and confocal microscopy. Techniques undergoing rapid development and with potential application to the mission of the U.S. Department of Energy’s Office of Biological and Environmental Research (BER) include X-ray free-electron laser ultrafast diffraction, time-resolved X-ray scattering, cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET), infrared imaging methods, super-resolution fluorescence imaging. New technologies not yet fully developed and applied to research supported by BER’s Biological Systems Science Division include dynamic EM, fluctuation scattering, ptychography, and in soil sensors. Key: Å, Angstrom; EXAFS, extended X-ray absorption fine structure; fs, femtosecond; ks, kilosecond; μm, micrometer; μs, microsecond; mm, millimeter; ms, millisecond; nm, nanometer; ns, nanosecond; ps, picosecond; s, second; SAXS, small-angle X-ray scattering; SEM, scanning electron microscopy; TEM, transmission electron microscopy; TIRF, total internal reflection fluorescence; USANS, ultrasmall-angle neutron scattering; WAXS, wide-angle X-ray scattering; XANES, X-ray absorption near edge structure.

Several challenges common to all the research themes emerged throughout workshop discussions. Translating information from genomic studies to the molecular and cellular realm for characterization will require increased throughput for existing technologies and the development of new high-throughput approaches. Achieving these goals will necessarily involve more automation and computational algorithms to manage the high data volumes that will be produced. Improved machine-learning approaches and large data-handling capacity will be essential. Integration of disparate data types from multiple and heterogeneous sources remains a challenge, so continued development of integrative and interpretive computational approaches is needed. Similar needs also were discussed at a workshop hosted by DOE’s Office of Advanced Scientific Computing Research (ASCR), the DOE Exascale Requirements Review, held March 28–31, 2016, in Rockville, Md., which generated the meeting report, ASCR Exascale Requirements Review

The tools and methods described in this BER report are critical for advancing the deep understanding of complex, multicomponent systems that are central to bioenergy and the environment. While new technologies are needed for advancing leading-edge biological insights, they are of limited value if they are not readily accessible by the scientists who need them to conduct their research. As new instruments, platforms, and approaches are created, it is important that they be developed in ways that ultimately enable biology researchers to use them, either by adopting them in their own laboratories or by having access to the tools, appropriate expertise, and support at national user facilities. Elements will include robust hardware, physiologically relevant sample preparation and measurement conditions, automation, sophisticated analytical algorithms, and user-friendly interfaces. For facility-based technologies, long-term and productive community access requires recognition of the need for ongoing operational support.

Described in the report are some of the workshop’s identified challenges to studying the biological systems of interest to BSSD, which has a history of developing and supporting highly sophisticated research tools and techniques and ensuring that researchers can access them to advance science in support of the division’s goals. Workshop discussions reflected in this document will help guide the next generation of imaging and analytical instrumentation needed to gain a predictive understanding of biological systems supporting DOE’s energy and environmental missions.