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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The Origins of Photosynthesis in a Sun-Loving Bacteria

Crystallization to Structural Solution
Crystallization to Structural Solution: (a) Emerald-colored protein crystals used. (b) Representative diffraction pattern with spots highlighted in blue circles and resolution rings designated by dashed lines. (c) Artistic representation developed from these patterns of the resultant three-dimensional protein crystal structure of the heliobacterial reaction center. [Courtesy Christopher Gisriel, Arizona State University]
Reaction centers (RCs) are pigment-protein complexes that drive photosynthesis by converting light into chemical energy (electrons) to power the cell, possibly arising from a homodimeric protein. The symmetry of a homodimer is broken in heterodimeric rRC structures, while the 2.2-Angstrom resolution X-ray structure of the homodimeric RC-photosystem from the phototroph Heliobacterium modesticaldum exhibits perfect C2 symmetry. The core polypeptide dimer and two small subunits coordinate 54 bacteriochlorophylls and 2 carotenoids that capture and transfer energy to the electron-transfer chain at the center, which performs charge separation and comprises 6 (bacterio)chlorophylls and an iron-sulfur cluster; unlike other RCs, it lacks a bound quinone. This structure preserves characteristics of the ancestral reaction center, providing insight into the evolution of photosynthesis.

Enough sunlight hits the Earth to power the planet many times over—if energy capture were more efficient. Thus, scientists have looked at nature’s means to understand photosynthesis in a sun-loving, soil-dwelling bacterium. A team of scientists has gained a fundamental understanding of the inner workings of this vital process and how it differs among plant systems. Their discovery may provide a new template for beginning organic-based solar panel design (i.e., “artificial leaves”) or possibly renewable biofuel applications.

But the team’s structural biologists are capturing freeze-frame images of crystallized proteins throughout the whole process of the simplest form of photosynthesis, in the single-celled heliobacteria, which are fundamentally different than plants. Instead of using water like plants, for example, heliobacteria use hydrogen sulfide and grow without oxygen; after photosynthesis, they give off the foul-smelling sulfur gas in place of oxygen. The scientists grew the “perfect” X-ray diffracting crystal charge after many initial trials. In this breakthrough study, they found an almost perfect symmetry in the heliobacter RC, which helps it gather every available photon of near-infrared light to build chlorophylls.

Gisriel, C., et al. “Structure of a symmetric photosynthetic reaction center–photosystem,” Science 357(6355), 1021–1025 (2017). [DOI:10.1126/science.aan5611].

Instruments and Facilities Used: X-ray macromolecular crystallograpy at Biodesign Center for Applied Structural Discovery at Arizona State University. Advanced Light Source at Berkeley Center for Structural Biology at Lawrence Berkeley National Laboratory. Structural Biology Center–CAT beamline of the Advanced Photon Source at Argonne National Laboratory.

Exploring the Roots of Photosynthesis in a Soil-Dwelling Bacterium

structure H. modesticaldum
Overall structure of the H. modesticaldum photosynthetic reaction center. The transmembrane helices are shown in gold and red. Light-harvesting “antenna” molecules are colored green, and the electron-transfer chain is colored blue. [From Gisriel, C., et al. “Structure of a Symmetric Photosynthetic Reaction Center–Photosystem,” Science 357(6355), 1021–1025 (2017). DOI:10.1126/science.aan5611. Reprinted with permission from AAAS.]
The simplest known bacterium able to drive photosynthesis is found in muddy soils near hot springs. Heliobacterium modesticaldum is a sun-loving, soil-dwelling, thermophilic bacterium that photosynthesizes near-infrared light, unlike plants, which use different parts of visible light. The photosynthesis reaction centers (RCs) of H. modesticaldum are thought to resemble the earliest common ancestor of all photosynthesis complexes, which evolved around three billion years ago. Thus, a clear, detailed picture of the H. modesticaldum RC would provide valuable insight into the early evolution of photosynthesis. However, successfully purifying an RC protein and growing the crystals needed for X-ray crystallography can be a lengthy, difficult process.

Researchers successfully capped 7 years of work by obtaining a high-resolution (2.2 Å) structure of the membrane protein at the heart of the photosynthetic RC of H. modesticaldum. The structure revealed details such as the interactions between light-harvesting “antenna” molecules and the electron-transfer chain, where the initial steps required to convert photon energy into chemical energy occurs. With this data, the research team was able to make comparisons to other types of RCs, gaining new perspectives on the early evolution of photosynthesis and how nature optimized light-driven energy collection. This work will help unlock the secrets of photosynthesis, possibly leading to the development of cleaner, solar-based renewable energy.

Gisriel, C., et al. “Structure of a Symmetric Photosynthetic Reaction Center–Photosystem.” Science 357(6355), 1021–1025 (2017). [DOI:10.1126/science.aan5611].

Instruments and Facilities: Beamline 8.2.1 at Advanced Light Source at Lawrence Berkeley National Laboratory; Advanced Photon Source at Argonne National Laboratory.

X-ray Footprinting Solves Mystery of Metal-Breathing Protein

3D structural rendering of protein
Results from X-ray footprinting mass spectrometry (XFMS) experiments at Lawrence Berkeley National Laboratory’s (LBNL) Advanced Light Source, mapped out on these three-dimensional structural renderings of a protein, helped researchers identify where the protein binds with a mineral. Red areas indicate possible binding areas. [Reprinted with permission from Fukushima, T., et al. “The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface.” J. Am. Chem. Soc. 139(36), 12647–12654, (2017). [DOI:10.1021/jacs.7b06560]. Copyright (2017) American Chemical Society.]
Scientists have discovered the details of an unconventional coupling between a bacterial protein and a mineral that allows the bacterium to breathe when oxygen is not available. This research could lead to new innovations in linking proteins to other materials for bio-based electronic devices, such as sensors that can diagnose disease or detect contaminants. It also could help researchers to understand and control the chemical reactions sparked by these protein-material interactions and, eventually, how organisms remodel their environment and make biominerals.

Researchers relied on an X-ray-based technique known as “footprinting” to pinpoint the chemical connections between the bacterial protein and nanoparticles composed of iron and oxygen. The study identified a previously mapped, surprisingly small and weak binding site, but unknown was how the site bound to the metal-containing mineral because conventional techniques can’t see this binding process. Footprinting reveals interactions of proteins in a near-native environment. The protein selected for the study is from the metal-reducing bacterium Shewanella oneidensis, which “eats sugar and basically breathes minerals” when oxygen is unavailable. This study already is providing ideas on how to redesign these proteins to make better electronic connections and thus more sensitive bioelectronic sensors.

Fukushima, T., et al. “The Molecular Basis for Binding of an Electron Transfer Protein to a Metal Oxide Surface.” J. Am. Chem. Soc. 139(36), 12647–12654, (2017). [DOI:10.1021/jacs.7b06560].

Instruments and Facilities Used: X-ray beamline 5.3.1 of the Advanced Light Source (ALS) to perform “footprinting” and mass spectrometry at the Biological Nanostructures Facility at the Molecular Foundry, both at Lawrence Berkeley National Laboratory (LBNL), and the LBNL-led DOE Joint BioEnergy Institute.

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The CRISPR Target-Recognition Mechanism

Cas1-Cas2 complexSurface representation of the Cas1-Cas2 complex, consisting of four Cas1 proteins (light and dark green) and two Cas2 proteins (yellow). Donor DNA (brown) is being integrated into the target DNA (blue), at a precise location in the CRISPR array, following a short leader sequence (red). [From Wright, A. V., et al.  “Structures of the CRISPR Genome Integration Complex,” Science 357(6356), 1113–1118 (2017). [DOI:10.1126/science.aao0679. Reprinted with permission from AAAS*.]

* Readers may view, browse, and/or download material for temporary copying purposes only, provided these uses are for noncommercial personal purposes. Except as provided by law, this material may not be further reproduced, distributed, transmitted, modified, adapted, performed, displayed, published, or sold in whole or in part, without prior written permission from the publisher.

 

 

Bacterial DNA is characterized by regions of clustered regularly interspaced short palindromic repeats (CRISPRs) and associated Cas proteins (CRISPR-associated endonucleases). The CRISPR-Cas system has revolutionized gene editing by vastly simplifying the insertion of short snippets of new (“donor”) DNA into very specific locations of target DNA. Researchers in this study have discovered how Cas proteins recognize their target locations with such great specificity. They used x-ray crystallography to solve the structures of Cas1 and Cas2—responsible for DNA-snippet capture and integration—as the proteins were bound to synthesized DNA strands designed to mimic different stages of the process. The research also demonstrated how the system works in its native context as part of a bacterial immune system and how Cas proteins act as general-purpose molecular recording devices—tools for encoding information in genomes.

Cas1 appears to have evolved from a more “promiscuous” (less selective) type of enzyme that catalyzes the movement of DNA sequences from one position to another (a transposase). At some point, Cas1 acquired an unusual degree of specificity for a particular location in the bacterial genome, the CRISPR array. This specificity is critical to the bacteria, both for acquiring immunity and for avoiding genome damage caused by the insertion of viral fragments at the wrong location. The researchers wanted to learn how Cas1-Cas2 proteins recognize the target sequence to enable comparison with previously studied transposases and integrases (i.e., enzymes that catalyze the integration of donor DNA into target DNA) and to determine whether the proteins can be altered to recognize new sequences for custom applications.

The researchers crystallized Cas1-Cas2 in complex with preformed DNA strands that mimicked reaction intermediates and products. X-ray crystallography revealed that the structures showed substantial distortions in the target DNA, but there were surprisingly few sequence-specific contacts with the Cas1-Cas2 complex, and the DNA’s resulting flexibility produced disorder in the crystals. Attempts to model the DNA across the disordered sections showed that the DNA had to be even more distorted. Cryoelectron microscopy experiments, coupled with the crystallography data, confirmed that an accessor protein called the integration host factor (IHF) introduces an additional sharp bend in the DNA, bringing an upstream recognition sequence into contact with Cas1 to increase both the specificity and efficiency of integration. The architecture of the CRISPR integration complex suggests that subtle adjustment of the distance between Cas1 active sites could reprogram the system to recognize different target sites. Changes in its architecture could be exploited, thereby, for genome tagging applications and also may explain the natural divergence of CRISPR arrays in bacteria.

Wright, A. V., et al. “Structures of the CRISPR Genome Integration Complex,” Science 357(6356), 1113–1118 (2017). [DOI:10.1126/science.aao0679].

Instruments and Facilities Used: X-ray macromolecular crystallography; beamline 8.3.1; protein crystallography (PX); and scattering/diffraction at the Advanced Light Source at Lawrence Berkeley National Laboratory; Stanford Synchrotron Radiation Light Source 9-2 beamline.

 

Novel Orange Carotenoid Proteins Shedding Light on Evolution of Cyanobacteria Photoprotection

blue-green algae
Filamentous blue-green algae [Courtesy Landcare Research. Reused under a Creative Commons license (CC By 4.0, https://creativecommons.org/licenses/by/4.0/).]
A research study has identified and characterized a new, functionally distinct member of the orange carotenoid protein (OCP) family. The OCP complex enables chromatically acclimating blue-green algae to avoid cellular damage and growth inhibition in conditions of high light or nutrient stress. In a recent bioinformatic analysis of all available cyanobacterial genomes, the group found that many of these ecophysiologically diverse organisms encode more than one copy of the full-length OCP. The study’s focus was the filamentous blue-green algae Tolypothrix, which encodes two OCPs.

One copy was determined to be functionally equivalent to the well-characterized OCP of Synechocystis cyanobacteria, dubbed OCP1. But the second, OCP2, was distinct in several key aspects. The researchers hypothesize that OCP2 and another bioinformatically identified protein, OCPx, reflect intermediate stages in the evolution of photoprotection in cyanobacteria.

Bao, H., et al. “Additional Families of Orange Carotenoid Proteins in the Photoprotective System of Cyanobacteria.” Nature Plants 3, Article 17089 (2017). [DOI:10.1038/nplants.2017.89].

Instruments and Facilities: X-ray macromolecular crystallography and diffraction at Advanced Light Source at Lawrence Berkeley National Laboratory.

Study on Human Skin Microbiome Finds Archaea Abundance Associated with Age

archaeal cells
(Left, blue and green images) Fluorescence images of archaeal cells in skin wipe samples; the Advanced Light Source (ALS) at Argonne National Laboratory was used to measure infrared absorption spectra of different Archaea types. (Right, illustration) The hierachical chart of the human skin archaeome with Thaumarchaeota (red), Euryarchaeota (green), and Crenarchaeota (blue). [From Moissl-Eichinger, C., et al. “Human Age and Skin Physiology Shape Diversity and Abundance of Archaea on Skin.” Sci. Rep. 7, 4039 (2017). [DOI:10.1038/s41598-017-04197-4]. Reused under a Creative Commons license (CC BY 4.0, https://creativecommons.org/licenses/by/4.0/).]
What’s on your skin? Your skin is crawling with single-celled microorganisms—and they’re not just bacteria. This study found that the skin microbiome also contains the extreme-loving archaea, and that the amount of these microbes varies with age. The microbiome forms a protective layer that protects the body from pathogens. Both genetic and chemical analyses of samples collected from human volunteers ranging in age from 1 to 75 showed that archaea were most abundant in subjects younger than 12 and older than 60. Before the study, the existence of archaea on human skin was unknown, but they are now known to play an important part. The international study also determined that gender was not a factor but that people with dry skin have more archaea. Results from genetic analysis and infrared spectroscopy imaging allowed scientists to link lower levels of oily secretion of sebaceous glands and with increased archaea, most of which have proven to be beneficial.

Moissl-Eichinger, C., et al. “Human Age and Skin Physiology Shape Diversity and Abundance of Archaea on Skin.” Sci. Rep. 7, 4039 (2017). [DOI:10.1038/s41598-017-04197-4].

Instruments and Facilities Used: Fluorescence in situ hybridization (FISH); quantitative polymerase chain reaction (PCR); next-generation sequencing; Fourier Transform infrared (FTIR) focal plan array (FPA) hyperspectral imaging. Facilities: Advanced Light Source at the Berkeley Synchrotron Infrared Structural Biology Imaging Project; BioTechMed-Graz, the Bavaria California Technology Center.

Could This Enzyme Help Turn Biofuel Waste into Something Useful?

LigM Structure
The protein structure of LigM was determined using X-ray crystallography, revealing novel structural elements that are unique to LigM (red) in addition to a conserved tetrahydrofolate-binding domain (gray) that is found throughout life. LigM binds to its substrates (green) using internal binding cavities. [From Kohlera, A. C., et al. “Structure of Aryl O-Demethylase Offers Molecular Insight into a Catalytic Tyrosine-Dependent Mechanism.” PNAS 114(16), E3205–E3214 (2017). DOI:10.1073/pnas.1619263114.]
Joint BioEnergy Institute study targets LigM for its role in breaking down aromatic pollutants

A protein used by common soil bacteria is providing new clues in the effort to convert aryl compounds, a common waste product from industrial and agricultural practices, into something of value. The protein structure of the enzyme LigM was determined using X-ray crystallography, revealing novel structural elements (red in figure) and a conserved tetrahydrofolate-binding domain (gray), with LigM binding to its substrates (green) using internal binding cavities.

Researchers used have resolved the soil bacterium Sphingomonas to metabolize aryl compounds derived from lignin, the stiff, organic material that gives plants their structure. In biofuel production, aryl compounds are a byproduct of the breakdown of lignin, some pathways of which involve demethylation, an often critical precursor to additional modification steps of lignin-derived aryl compounds. The simple, single-enzyme system of LigM, as well as its functionality over a broad temperature range, makes it an attractive demethylase for use in aromatic conversion. Other findings included: half the LigM enzyme was homologous to known structures with a tetrahydrofolate-binding domain that is found in both simple and complex organisms; the other half of LigM’s structure is completely unique, providing a starting point for determining where its aryl substrate-binding site is located; and LigM is a tyrosine-dependent demethylase. This research provides groundwork needed to aid in developing an enzyme-based system for converting aromatic waste into useful products.

Kohlera, A. C., et al. “Structure of Aryl O-Demethylase Offers Molecular Insight into a Catalytic Tyrosine-Dependent Mechanism.” PNAS 114(16), E3205–E3214 (2017). [DOI:10.1073/pnas.1619263114].

Instruments and Facilities Used: Beam line 8.2.2 and X-ray macromolecular crystallography at Berkeley Center for Structural Biology Advanced Light Source at Lawrence Berkeley National Laboratory.

Sequencing of Green Alga Genome Provides Blueprint to Advance Clean Energy, Bioproducts

Chromoshloris zofingiensis
Chromoshloris zofingiensis cell morphology. Cryo-soft X-ray tomography of a reconstructed cell with segmented nucleus (purple), chloroplast (green), mitochondria (red), lipids (yellow), and starch granules within the chloroplast (blue). (A) A representative orthoslice of the reconstructed cell. (B) Three-dimensional segmentation over two orthogonal orthoslices. (C) Segmented chloroplast and nucleus. (D) Fully segmented cell. [Reprinted with permission from Roth, M. S., et al. “Chromosome-Level Genome Assembly and Transcriptome of the Green Alga Chromochloris zofingiensis Illuminates Astaxanthin Production.” PNAS 114(21), E4296–E4305 (2017). [DOI:10.1073/pnas.1619928114]. Image credit Melissa S. Roth, HHMI/UC-Berkely and Andreas Walter, LBNL]
Microalgae have potential to help meet energy and food demands without exacerbating environmental problems. The unicellular green alga Chromochloris zofingiensis produces lipids for biofuels and a highly valuable carotenoid nutraceutical, astaxanthin. Thus advanced understanding of its biology is needed to facilitate commercial development. The assembly of the C. zofingiensis chromosome-level nuclear genome, organelle genomes, and transcriptome from diverse growth conditions was derived from a combination of short- and long-read sequencing in conjunction with optical mapping, revealing a compact genome of ∼58 Mbp distributed over 19 chromosomes containing 15,274 predicted protein-coding genes. Found in the genome were 2 genes encoding beta-ketolase (BKT), the key enzyme synthesizing astaxanthin, and both were up-regulated by high light. Isolation and molecular analysis of astaxanthin-deficient mutants. Moreover, the transcriptome under high light exposure revealed candidate genes that could be involved in critical yet missing steps of astaxanthin biosynthesis, including ABC transporters, cytochrome P450 enzymes, and an acyltransferase. The high-quality genome and transcriptome provide insight into the green algal lineage and carotenoid production. Microalgae are a promising source of sustainable bioproducts for the increasing demand for food and energy without exacerbating worsening environmental problems. The algae have potential for use as a biofuel feedstock and nutraceutical molecules, including the carotenoid astaxanthin. Analyses of the C. zofingiensis genome and transcriptome and experiments characterizing astaxanthin production advance understanding of algae and carotenoids  and enhance the commercial potential of C. zofingiensis.

Roth, M. S., et al.. “Chromosome-Level Genome Assembly and Transcriptome of the Green Alga Chromochloris zofingiensis Illuminates Astaxanthin Production.” PNAS 114(21), E4296–E4305 (2017). [DOI:10.1073/pnas.1619928114].

Instruments and Facilities Used: Soft X-ray tomography at National Center for X-ray Tomography (NCXT), operated jointly by Berkeley Lab (LBNL) and University of California, San Francisco, at LBNL’s Advanced Light Source. Other techniques: whole-genome optical mapping, high light RNA sequencing, transcriptome sequencing, and long read sequencing.