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.
Scientists in this study have successfully demonstrated the ability to manipulate the neutron contrast of detergent micelles by incorporating a similar detergent with deuterium-labelled alkyl chains. The presence of excess detergent micelle scattering often has a detrimental influence on scattering data obtained for membrane protein–detergent complexes. Isolation of the scattering signal from the protein of interest can be accomplished by eliminating all scattering from the detergent. Using this approach enabled determination of the overall structure and oligomeric state of a small membrane protein enzyme.
Oliver, R. C., et al. “Designing Mixed Detergent Micelles for Uniform Neutron Contrast.” The Journal of Physical Chemistry Letters8(20), 5041–5046 (2017). [DOI:10.1021/acs.jpclett.7b02149].
Instruments and Facilities Used: Small angle neutron scattering (SANS): Bio-SANS beamline (CG3) of the High-Flux Isotope Reactor at Oak Ridge National Laboratory (ORNL). Recorded scattering data using MantidPlot software. Neutron contrast studies: ModULes for the Analysis of Contrast (MULCh) Variation Data at University of Sydney (smb-research.smb.usyd.edu.au/NCVWeb/).
Funding Acknowledgements: Neutron scattering studies at CG-3 Bio-SANS instrument at the High-Flux Isotope Reactor (HFIR), Oak Ridge National Laboratory (ORNL), sponsored by the Office of Biological and Environmental Research (OBER) and Office of Basic Energy Sciences (OBES) Scientific User Facilities Division (SUF), U.S. Department of Energy (DOE) Office of Science. Work benefited from use of the SasView application, originally developed under National Science Foundation (NSF) Award DMR-0520547. SasView contains code developed with funding from the European Union’s Horizon 2020 Research and Innovation Programme under the SINE2020 project, Grant Agreement No 654000. Manuscript authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with DOE. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/doe-public-access-plan).
Biomass pretreatment is necessary to make cellulose accessible to hydrolysis for conversion to biofuels. Understanding water’s role in cellulose reactivity will aid discovery of the underlying processes that change biomass morphology and reactivity during different pretreatment regimes for biofuels production. In this study, cellulose-water interactions were examined using neutron scattering supported by molecular dynamics simulation. The data show two distinct populations of water molecules—one tightly “bound” to the surface and the other interfibrillar and translationally mobile. Accurate models of hydration water in the cell wall can address fundamental questions about cellulose-water interactions. The mobility of the interfibrillar water is also important to enzyme and chemical attack and is distinct from the bound and bulk water.
O’Neill, H. M., et al. “Dynamics of Water Bound to Crystalline Cellulose.” Sci. Rep.7, Article 11840 (2017). [DOI:10.1038/s41598-017-12035-w].
Instruments and Facilities Used: Deuterium labeling, neutron scattering, and molecular dynamics simulation; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer, at Oak Ridge National Laboratory (ORNL) Spallation Neutron Source (SNS). Structural characterization using small-angle neutron scattering (SANS) CG-3 Bio-SANS instrument at High Flux Isotope Reactor (HFIR) facility of ORNL and X-ray diffraction (XRD) analysis combined with SANS. Wide-Angle X-ray Diffraction (WAXD) using theta-theta goniometer Bruker D5005 instrument; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer at ORNL SNS; Molecular Dynamics (MD) Simulations (GROMACS software and the CHARMM C36 carbohydrate force field); and TIP4P water model.
Funding Acknowledgements: Deuterium labeling, neutron scattering, and molecular dynamics simulation; quasi-elastic neutron scattering (QENS) using BASIS, the Backscattering Spectrometer, at Oak Ridge National Laboratory (ORNL) Spallation Neutron Source (SNS). Structural characterization using small-angle neutron scattering (SANS) CG-3 Bio-SANS instrument at High Flux Isotope Reactor (HFIR) facility, ORNL, and x-ray diffraction (XRD) analysis combined with SANS. Wide-angle x-ray diffraction (WAXD) using theta-theta goniometer Bruker D5005 instrument; QENS; molecular dynamics (MD) simulations (GROMACS software and CHARMM C36 carbohydrate force field); and TIP4P water model. H.O’N., J.H., B.E., J.C.S., P.L. and B.H.D. support: U.S. Department of Energy (DOE) Genomic Science Program, Office of Biological and Environmental Research (OBER), DOE Office of Science, under Contract FWP ERKP752, for sample preparation and QENS studies. SANS studies on Bio-SANS by S.V.P. and V.U. support: OBER-funded Center for Structural Molecular Biology (CSMB) under Contract FWP ERKP291, using facilities supported by the Office of Basic Energy Sciences (OBES), DOE Office of Science. Molecular dynamics (MD) simulations performed by L.P. supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center, funded by DOE OBES, under Award DE-SC0001090. Research used resources of the National Energy Research Scientific Computing Center (NERSC), a DOE Office of Science User Facility supported under Contract No. DE-AC02-05CH11231. E.M. support: Oak Ridge National Laboratory’s (ORNL) Spallation Neutron Source (SNS), funded by the DOE OBES Scientific User Facilities Division. Manuscript authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with DOE.
A multimodal approach used in this study examined wood decay by the brown rot fungi, Gloeophyllum trabeum or Rhodonia placenta, that degrade wood using a chelator-mediated Fenton (CMF) reaction. Small-angle neutron scattering (SANS) showed changes in microfibril bundling and lignin structure during biomass breakdown. Complementary approaches, sum frequency generation (SFG) spectroscopy, X-ray diffraction, atomic force microscopy (AFM), and transmission electron microscopy (TEM) also contributed information on nanoscale structural changes in wood over time. Woods studied were southern yellow pine (Pinus spp.) and birch (Betula verrucosa Ehr.). The data support a degradation mechanism in which sugars released by non-enzymatic action diffuse from the cell wall, facilitated by increasing the porosity of the cell walls. This is a paradigm shift in understanding the mechanism of brown rot fungal degradation.
Goodell, B., et al. “Modification of the Nanostructure of Lignocellulose Cell Walls via a Non-Enzymatic Lignocellulose Deconstruction System in Brown Rot Wood-Decay Fungi.” Biotechnol. Biofuels10(1), 179 (2017). [DOI:10.1186/s13068-017-0865-2].
Instruments and Facilities Used: Small angle neutron scattering (SANS) – Bio-SANS at High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory (ORNL). Sum frequency generation (SFG) spectroscopy, broadband SFG system. Chelator-mediated Fenton treatments (CMF) and cellulase treatment. X-ray diffraction analysis (XRD) – PANalytical Empyrean diffractometer, The Netherlands, equipped with a Cu X-ray source. Attenuated total reflectance Fourier transform infrared analysis (ATR-FTIR): Nicolet 8700 FTIR Spectrometer (Thermo Scientific) equipped with a smart iTR diamond ATR unit, a KBr beam splitter, and a deuterated triglycine sulfate (DTGS) detector. Atomic force microscopy (AFM) of brown-rotted wood surfaces. Nanoscope IIIa AFM-Digital Instruments, Santa Barbara, California with three 5 µm scans. Transmission electron microscopy (TEM) – Philips CM12 TEM instrument (Philips, Eindhoven, The Netherlands); images recorded on Kodak 4489 negative film and the films subsequently scanned using an Epson Perfection Pro 750 film scanner.
Funding Acknowledgements: Dr. Zhu support: Chinese Forestry Industry Research Special Funds for Public Welfare Projects (#201204702-B2). Eastwood support: UK Natural Environment Research Council, award #NE/K011588/1. Daniel support: Formas Grant 2015-469. Support from ORNL-Proposal IPTS-12345/CG-3. Sum frequency generation (SFG) spectroscopy, x-ray diffraction (XRD), and infrared (IR) studies support: Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center, funded by the Office of Basic Energy Sciences (OBES), U.S. Department of Energy (DOE) Office of Science, under Award Number DE-SC0001090. Pingali and O’Neill support: Biofuels SFA funded by the DOE Genomic Science Program, Office of Biological and Environmental Research (OBER), under Contract FWP ERKP752. Bio-SANS support: Center for Structural Molecular Biology, supported by DOE OBER under Contract FWP ERKP291. Neutron scattering facilities at Oak Ridge National Laboratory (ORNL) support: Scientific User Facilities Division, DOE OBES. Research also supported by U.S. Department of Agriculture’s (USDA) National Institute of Food and Agriculture (NIFA) and University of Massachusetts Amherst’s Center for Agriculture, Food and the Environment and the Microbiology Department: project # MAS00511.
Many unanswered questions remain about how macromolecules in solution perturb the water molecules in which they are immersed. Scientists used neutron scattering to provide an experimental description of the dynamical perturbation of water molecules surrounding proteins in solution, which play an important role in protein function and stability. Quantifying the magnitude of the perturbation of water around the green fluorescent protein (GFP), they found a systematic length-scale dependence of the dynamical retardation factor compared to the perturbation of bulk water. The findings deepen the understanding of water perturbation, which is of practical interest to researchers in food science, personal care, pharmaceutics, and protein dynamics.
Perticaroli, S., et al. “Description of Hydration Water in Protein (Green Fluorescent Protein) Solution.” J. Am. Chem. Soc.139(3), 1098–1105 (2017). [DOI:10.1021/jacs.6b08845].
Instruments and Facilities Used: Neutron scattering at Center for Structural Molecular Biology and Spallation Neutron Source at Oak Ridge National Laboratory.
Funding Acknowledgements: H.O’N. and Q.Z. support: Center for Structural Molecular Biology, funded by the Office of Biological and Environmental Research (OBER), U.S. Department of Energy (DOE) Office of Science, under Contract FWP ERKP291. Research at Oak Ridge National Laboratory’s (ORNL) Spallation Neutron Source (SNS) sponsored by the Office of Basic Energy Sciences (OBES) Scientific User Facilities Division, DOE Office of Science. ORNL facilities sponsored by UT-Battelle, LLC, for the DOE under Contract No. DEAC0500OR22725.
The plant cellulose synthesis complex is a large multi-subunit transmembrane protein complex responsible for synthesis of cellulose chains and their assembly into microfibrils. The image shows ab initio structures of CESA trimers calculated from small-angle scattering data represented by semi-transparent grey surface envelopes, superposed with the computational atomic models in orange. Image credits: Thomas Splettstoesser, scistyle.com, Berlin Germany
Vandavasi, V.G., D.K. Putnam, Q. Zhang, L. Petridis, W.T. Heller, B.T. Nixon, C.H. Haigler, U. Kalluri, L. Coates, and P. Langan. 2016. “A structural study of CESA1 catalytic domain of Arabidopsis cellulose synthesis complex: evidence for CESA trimers.” Plant physiology 170(1):123-35. [DOI: 10.1104/pp.15.01356]
Funding Acknowledgements: Molecular biology and structural characterization: H.O., V.G.V., Q.Z., W.T.H., L.P., U.K., J.C.S., P.L., and L.C., supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (ORNL), managed by UT-Battelle, LLC, for the U.S. Department of Energy (DOE) under Contract Number DE-AC05-00OR22725. Small-angle scattering (SAS) and computational analysis, performed by H.O., V.G.V., L.P., B.T.N., and C.H.H., supported by Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center (EFRC) funded by the Office of Basic Energy Sciences (OBES). DOE Office of Science. J.M. and D.K.P. support: High Performance Computing Grant from Oak Ridge Associated Universities (ORAU). Bio-SANS is operated by the Center for Structural Molecular Biology at ORNL, supported by Office of Biological and Environmental Research (OBER), DOE Office of Science, Project ERKP291. EQ-SANS at Spallation Neutron Source (SNS) and High Flux Isotope Reactor (HFIR) sponsored by OBES Scientific User Facilities Division, DOE Office of Science, at ORNL. Sai Venkatesh Pingali: assistance with the operation of Bio-SANS and data reduction. Paul Abraham and the BioEnergy Science Center (BESC) proteomics facilities: validation of purified proteins. BESC supported by DOE OBER. Mass spectrometry (MS) analysis carried out by DOE OBERsupported Bioenergy Research Center proteomics pipeline. Use of the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory (BNL), supported by OBES, DOE Office of Science, under Contract Number DE-AC02-98CH10886.