Nav: Home

New protein spin labelling technique

August 20, 2019

Site-directed spin labelling (SDSL) used in combination with electron paramagnetic resonance (EPR) spectroscopy has been a tried and trusted technique for elucidating the structure, function and dynamics of proteins and protein complexes. Nitroxide-based spin labels are among the most popular and best established ones because they are small, non-disturbing and exhibit excellent spectroscopic properties. "Ideal spin labelling procedures exhibit high reaction rates and selectivity", explains Professor Malte Drescher, Professor for Spectroscopy of Complex Systems at the University of Konstanz's Department of Chemistry and main author on the study alongside Professor Valentin Wittmann, who specializes in organic synthesis.

"Achieving high reactivity and high selectivity both at the same time can be a problem", continues Drescher. "Conventional spin labels based, for instance, on Gadolinium(III) or trityl, display either very broad spectra and low modulation depths or very narrow spectra that are unsuitable for the kinds of experiments that we want to conduct". A new study published by Drescher, Wittmann and their team of University of Konstanz chemists, which was published online in the journal ChemBioChem Communications on 14 August 2019, introduces a new approach for labelling proteins that features nitroxide-based spin labels and genetically encoded noncanonical amino acids (ncAAs) as targets for SDSL.

"Nitroxides provide ideal spectral width and access to dynamic information", says Anandi Kugele, a doctoral researcher at the Konstanz Research School Chemical Biology (KoRS-CB) and first author on the study, who received a prestigious travel grant from the National High Magnetic Field Laboratory to present the results at the 2019 Rocky Mountain Conference on Magnetic Resonance in Denver, Colorado (USA). "Traditional nitroxide-based labels have limited redox stability, which is a drawback for in-cell applications. The challenge for us was to increase nitroxide stability and thus to adapt nitroxide-based spin labels for future routine in vivo use". To that end, the researchers developed a new spin label that can be attached to proteins by means of inverse-electron-demand Diels-Alder (DAinv) cycloaddition to genetically encoded ncAAs, a method which has proven suitable to a broad range of in vitro and in vivo applications. To achieve nitroxide stability, the researchers further used a protection strategy based on photoremovable protecting groups, which are known to protect nitroxides and to release them as needed.

The new spin label - termed photoactivatable nitroxide for DAinv reaction, or PaNDA for short - is water soluble, EPR-active and deprotection-efficient as both in vitro and in lysate tests with the two model proteins green fluorescent protein (GFP) and Escherichia coli oxidoreductase thioredoxin (TRX), which is found in virtually all known organisms, suggest. "We do need to improve on the method used to deliver the PaNDA spin label to cells and to test labelling and deprotection efficiencies inside the cell, amongst other things", concludes Malte Drescher: "But our research clearly demonstrates that, in principle, the PaNDA label can be used for EPR measurements in challenging biological environments, including the inside of cells. Our tests with E. coli lysate are very promising in this respect. This will open up a whole new range of opportunities for the study of proteins by means of EPR spectroscopy".
-end-
Facts:
  • University of Konstanz researchers develop an innovative site-directed spin labelling (SDSL) approach based on genetically encoded noncanonical amino acids (ncAAs) amenable to inverse-electron-demand Diels-Alder (DAinv) cycloaddition as well as a new spin label, PaNDA

  • The strategy yields high reaction rates, high selectivity and the possibility to deprotect the nitroxide in Escherichia coli lysate

  • Original publication: Anandi Kugele, Bjarne Silkenath, Jakob Langer, Valentin Wittmann, Malte Drescher, Protein Spin Labeling with a Photocaged Nitroxide Using Diels-Alder Chemistry, ChemBioChem Communications, published online 14 August 2019, DOI: https://doi.org/10.1002/cbic.201900318

  • Supported by the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme and by the German Research Foundation (DFG, Collaborative Research Centre 969 "Chemical and Biological Principles of Cellular Proteostasis", project C03)
Note to editors:

An image is available for download here: https://cms.uni-konstanz.de/fileadmin/pi/fileserver/2019/Bilder/new_protein_spin.jpg Caption: The novel photoactivatable nitroxide for DAinv reaction spin label for proteins, PaNDA. It can be ligated to proteins through a DAinv cycloaddition to genetically encoded noncanonical amino acids.

Credit: Anandi Kugele

Contact:

University of Konstanz
Communications and Marketing
Phone: +49 7531 88-3603
Email: kum@uni-konstanz.de

University of Konstanz

Related Science Articles:

75 science societies urge the education department to base Title IX sexual harassment regulations on evidence and science
The American Educational Research Association (AERA) and the American Association for the Advancement of Science (AAAS) today led 75 scientific societies in submitting comments on the US Department of Education's proposed changes to Title IX regulations.
Science/Science Careers' survey ranks top biotech, biopharma, and pharma employers
The Science and Science Careers' 2018 annual Top Employers Survey polled employees in the biotechnology, biopharmaceutical, pharmaceutical, and related industries to determine the 20 best employers in these industries as well as their driving characteristics.
Science in the palm of your hand: How citizen science transforms passive learners
Citizen science projects can engage even children who previously were not interested in science.
Applied science may yield more translational research publications than basic science
While translational research can happen at any stage of the research process, a recent investigation of behavioral and social science research awards granted by the NIH between 2008 and 2014 revealed that applied science yielded a higher volume of translational research publications than basic science, according to a study published May 9, 2018 in the open-access journal PLOS ONE by Xueying Han from the Science and Technology Policy Institute, USA, and colleagues.
Prominent academics, including Salk's Thomas Albright, call for more science in forensic science
Six scientists who recently served on the National Commission on Forensic Science are calling on the scientific community at large to advocate for increased research and financial support of forensic science as well as the introduction of empirical testing requirements to ensure the validity of outcomes.
World Science Forum 2017 Jordan issues Science for Peace Declaration
On behalf of the coordinating organizations responsible for delivering the World Science Forum Jordan, the concluding Science for Peace Declaration issued at the Dead Sea represents a global call for action to science and society to build a future that promises greater equality, security and opportunity for all, and in which science plays an increasingly prominent role as an enabler of fair and sustainable development.
PETA science group promotes animal-free science at society of toxicology conference
The PETA International Science Consortium Ltd. is presenting two posters on animal-free methods for testing inhalation toxicity at the 56th annual Society of Toxicology (SOT) meeting March 12 to 16, 2017, in Baltimore, Maryland.
Citizen Science in the Digital Age: Rhetoric, Science and Public Engagement
James Wynn's timely investigation highlights scientific studies grounded in publicly gathered data and probes the rhetoric these studies employ.
Science/Science Careers' survey ranks top biotech, pharma, and biopharma employers
The Science and Science Careers' 2016 annual Top Employers Survey polled employees in the biotechnology, biopharmaceutical, pharmaceutical, and related industries to determine the 20 best employers in these industries as well as their driving characteristics.
Three natural science professors win TJ Park Science Fellowship
Professor Jung-Min Kee (Department of Chemistry, UNIST), Professor Kyudong Choi (Department of Mathematical Sciences, UNIST), and Professor Kwanpyo Kim (Department of Physics, UNIST) are the recipients of the Cheong-Am (TJ Park) Science Fellowship of the year 2016.
More Science News and Science Current Events

Top Science Podcasts

We have hand picked the top science podcasts of 2019.
Now Playing: TED Radio Hour

Risk
Why do we revere risk-takers, even when their actions terrify us? Why are some better at taking risks than others? This hour, TED speakers explore the alluring, dangerous, and calculated sides of risk. Guests include professional rock climber Alex Honnold, economist Mariana Mazzucato, psychology researcher Kashfia Rahman, structural engineer and bridge designer Ian Firth, and risk intelligence expert Dylan Evans.
Now Playing: Science for the People

#540 Specialize? Or Generalize?
Ever been called a "jack of all trades, master of none"? The world loves to elevate specialists, people who drill deep into a single topic. Those people are great. But there's a place for generalists too, argues David Epstein. Jacks of all trades are often more successful than specialists. And he's got science to back it up. We talk with Epstein about his latest book, "Range: Why Generalists Triumph in a Specialized World".
Now Playing: Radiolab

Dolly Parton's America: Neon Moss
Today on Radiolab, we're bringing you the fourth episode of Jad's special series, Dolly Parton's America. In this episode, Jad goes back up the mountain to visit Dolly's actual Tennessee mountain home, where she tells stories about her first trips out of the holler. Back on the mountaintop, standing under the rain by the Little Pigeon River, the trip triggers memories of Jad's first visit to his father's childhood home, and opens the gateway to dizzying stories of music and migration. Support Radiolab today at Radiolab.org/donate.