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-funded Energy Frontier Research Center based at Washington University. Small angle neutron scattering (SANS) Bio-SANS instrument, beamline CG‑3, at Oak Ridge National Laboratory’s High Flux Isotope Reactor.

Precise Control of Neutron Contrast in Surfactant Micelles Provides Platform for Membrane Structure Studies

Detergent Micelles
The scattering collected for detergent at its solution match point, where contrast still persists between core and shell, produces a non-flat scattering profile (red). Incorporating the non-ionic detergent DDM with deuterium-labelled chains allows matching of the core and shell contrast, producing the flat scattering profile shown in blue. [Reprinted with permission from Oliver, R. C., S. V. Pingali, and V. S. Urban. “Designing Mixed Detergent Micelles for Uniform Neutron Contrast.” J. Phys. Chem. Lett. 8, 5041–5046 (2017). [DOI: 10.1021/acs.jpclett.7b02149]. Copyright 2017 American Chemical Society.]
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 Letters 8(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 (

Dynamics on Cellulose Show Two Important Populations from Neutron Scattering and Simulations

Elastic Intensity Scans
Elastic intensity scans of dry and hydrated cellulose. Dashed lines denote inflection points in the curves at 220 and 260 K, the temperatures at which the surface water (nonfreezing) and interfibrillar water (freezing) become mobile in the hydrated cellulose sample, respectively. Inset illustration depicts water populations associated with cellulose. [Adapted from O’Neill, H., et al. “Dynamics of Water Bound to Crystalline Cellulose.” Sci. Rep. 7, 11840 (2017). [DOI:10.1038/s41598-017-12035-w]. Reused under a Creative Commons license (CC BY 4.0, Curve colors were modified, and additional labels and inset added.]
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.

Brown Rot Fungi Reveal a New Approach for Biomass Conversion to Fuels and Chemicals

Brown rot fungi

(A) Brown rot fungi mushrooms, (B) SANS profiles, (C) SFG spectra of brown rot fungi–mediated cellulose deconstruction, and (D) AFM images of repolymerized lignin in brown rot cell walls. [(A) Wikimedia Commons, Zinnmann; (B) Authors; (C) and (D) From 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. Biofuels 10, 179 (2017). [DOI 10.1186/s13068-017-0865-2]. Reused under a Creative Commons license (CC BY 4.0,]
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. Biofuels 10(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.

Description of Hydration Water in Green Fluorescent Protein Solution

green fluorescent protein
Graphic representation of hydration water surrounding green fluorescent protein. [Reprinted with permission from 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]. Copyright 2016 American Chemical Society.]
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.

Cellulose Synthesis Complex

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,, 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]