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PubMed | CNRS Institute of Pharmacology and Structural Biology, The Scintillon Institute, University Paris - Sud and European Synchrotron Radiation Facility
Type: Journal Article | Journal: Acta crystallographica. Section D, Structural biology | Year: 2016

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.


Powers M.L.,Monterey Bay Aquarium Research Institute | Powers M.L.,University of California at Santa Cruz | McDermott A.G.,Monterey Bay Aquarium Research Institute | Shaner N.C.,The Scintillon Institute | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2013

Calcium-binding photoproteins have been discovered in a variety of luminous marine organisms [1]. Recent interest in photoproteins from the phylum Ctenophora has stemmed from cloning and expression of several photoproteins from this group [2-5]. Additional characterization has revealed unique biochemical properties found only in ctenophore photoproteins, such as inactivation by light. Here we report the cloning, expression, and characterization of the photoprotein responsible for luminescence in the deep-sea ctenophore Bathocyroe fosteri. This animal was of particular interest due to the unique broad color spectrum observed in live specimens [6]. Full-length sequences were identified by BLAST searches of known photoprotein sequences against Bathocyroe transcripts obtained from 454 sequencing. Recombinantly expressed Bathocyroe photoprotein (BfosPP) displayed an optimal coelenterazine-loading pH of 8.5, and produced calcium-triggered luminescence with peak wavelengths closely matching the 493. nm peak observed in the spectrum of live B. fosteri specimens. Luminescence from recombinant BfosPP was inactivated most efficiently by UV and blue light. Primary structure alignment of BfosPP with other characterized photoproteins showed very strong sequence similarity to other ctenophore photoproteins and conservation of EF-hand motifs. Both alignment and structural prediction data provide more insight into the formation of the coelenterazine-binding domain and the probable mechanism of photoinactivation. © 2012 Elsevier Inc.


Francis W.R.,Monterey Bay Aquarium Research Institute | Francis W.R.,University of California at Santa Cruz | Christianson L.M.,Monterey Bay Aquarium Research Institute | Kiko R.,Leibniz Institute of Marine Science | And 4 more authors.
BMC Genomics | Year: 2013

Background: The lack of genomic resources can present challenges for studies of non-model organisms. Transcriptome sequencing offers an attractive method to gather information about genes and gene expression without the need for a reference genome. However, it is unclear what sequencing depth is adequate to assemble the transcriptome de novo for these purposes.Results: We assembled transcriptomes of animals from six different phyla (Annelids, Arthropods, Chordates, Cnidarians, Ctenophores, and Molluscs) at regular increments of reads using Velvet/Oases and Trinity to determine how read count affects the assembly. This included an assembly of mouse heart reads because we could compare those against the reference genome that is available. We found qualitative differences in the assemblies of whole-animals versus tissues. With increasing reads, whole-animal assemblies show rapid increase of transcripts and discovery of conserved genes, while single-tissue assemblies show a slower discovery of conserved genes though the assembled transcripts were often longer. A deeper examination of the mouse assemblies shows that with more reads, assembly errors become more frequent but such errors can be mitigated with more stringent assembly parameters.Conclusions: These assembly trends suggest that representative assemblies are generated with as few as 20 million reads for tissue samples and 30 million reads for whole-animals for RNA-level coverage. These depths provide a good balance between coverage and noise. Beyond 60 million reads, the discovery of new genes is low and sequencing errors of highly-expressed genes are likely to accumulate. Finally, siphonophores (polymorphic Cnidarians) are an exception and possibly require alternate assembly strategies. © 2013 Francis et al.; licensee BioMed Central Ltd.


Nakamura T.,Sanford Burnham Institute for Medical Research | Nakamura T.,The Scintillon Institute | Lipton S.A.,Sanford Burnham Institute for Medical Research | Lipton S.A.,University of California at San Diego | Lipton S.A.,The Scintillon Institute
Neurochemical Research | Year: 2016

Reactive nitrogen species, such as nitric oxide (NO), exert their biological activity in large part through post-translational modification of cysteine residues, forming S-nitrosothiols. This chemical reaction proceeds via a process that we and our colleagues have termed protein S-nitrosylation. Under conditions of normal NO production, S-nitrosylation regulates the activity of many normal proteins. However, in degenerative conditions characterized by nitrosative stress, increased levels of NO lead to aberrant S-nitrosylation that contributes to the pathology of the disease. Thus, S-nitrosylation has been implicated in a wide range of cellular mechanisms, including mitochondrial function, proteostasis, transcriptional regulation, synaptic activity, and cell survival. In recent years, the research area of protein S-nitrosylation has become prominent due to improvements in the detection systems as well as the demonstration that protein S-nitrosylation plays a critical role in the pathogenesis of neurodegenerative and other neurological disorders. To further promote our understanding of how protein S-nitrosylation affects cellular systems, guidelines for the design and conduct of research on S-nitrosylated (or SNO-)proteins would be highly desirable, especially for those newly entering the field. In this review article, we provide a strategic overview of designing experimental approaches to study protein S-nitrosylation. We specifically focus on methods that can provide critical data to demonstrate that an S-nitrosylated protein plays a (patho-)physiologically-relevant role in a biological process. Hence, the implementation of the approaches described herein will contribute to further advancement of the study of S-nitrosylated proteins, not only in neuroscience but also in other research fields. © 2015, Springer Science+Business Media New York.


PubMed | The Scintillon Institute
Type: Journal Article | Journal: Nature methods | Year: 2013

We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.


PubMed | The Scintillon Institute
Type: | Journal: Methods in cell biology | Year: 2014

More than 20 years after their discovery, fluorescent proteins (FPs) continue to be the subject of massive engineering efforts yielding continued improvements. Among these efforts are many aspects that should be of great interest to quantitative imaging users. With new variants frequently introduced into the research community, tried and true FPs that have been relied on for many years may now be due for upgrades to more modern variants. However, the dizzying array of FPs now available can make the initial act of narrowing down the potential choices an intimidating prospect. This chapter describes the FP properties that most strongly impact their performance in quantitative imaging experiments, along with their physical origins as they are currently understood. A workflow for evaluating a given FP in the researchers chosen experimental system (e.g., a specific cell line) is described.


PubMed | University of California at San Diego, The Scintillon Institute and Beckman Laser Institute
Type: | Journal: Scientific reports | Year: 2016

Hydrogen sulfide is a highly toxic gas-second only to carbon monoxide as a cause of inhalational deaths. Its mechanism of toxicity is only partially known, and no specific therapy exists for sulfide poisoning. We show in several cell types, including human inducible pluripotent stem cell (hiPSC)-derived neurons, that sulfide inhibited complex IV of the mitochondrial respiratory chain and induced apoptosis. Sulfide increased hydroxyl radical production in isolated mouse heart mitochondria and F2-isoprostanes in brains and hearts of mice. The vitamin B12 analog cobinamide reversed the cellular toxicity of sulfide, and rescued Drosophila melanogaster and mice from lethal exposures of hydrogen sulfide gas. Cobinamide worked through two distinct mechanisms: direct reversal of complex IV inhibition and neutralization of sulfide-generated reactive oxygen species. We conclude that sulfide produces a high degree of oxidative stress in cells and tissues, and that cobinamide has promise as a first specific treatment for sulfide poisoning.

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