Protein labeling is used by researchers in a variety of fields to help them understand how these important molecules affect the normal functioning of cells. Currently, proteins are labeled for study simply by fusing them to other fluorescent proteins, which allows researchers to use microscopy to track their movements through a cell. This approach has several drawbacks, however, not least being that the fluorescent proteins are often large enough to affect the function of the protein of interest.
Dr. Alex Deiters, associate professor of chemistry, along with colleague Dr. Jason Chin of the Laboratory of Molecular Biology at the Medical Research Council in Cambridge, U.K., have developed a way to attach a fluorophore – a fluorescent molecule about 20 times smaller than the fluorescent proteins currently in use – to a protein that is expressed in a mammalian cell.
Deiters and Chin developed a special 21st amino acid that they added to cells that were specially engineered to incorporate this amino acid into the protein they wanted to study (there are normally only 20 amino acids). This 21st amino acid has a "chemical handle" that only reacts with a specifically designed fluorophore, but not any cellular components. According to Deiters, "The reaction between the modified protein and the fluorophore is extremely fast, high yielding, and generates a stable link between both reaction partners. This novel methodology enables future cell biological studies that were previously not possible."
The research appears in the Feb. 5 issue of Nature Chemistry.
"We found that our approach gave us a higher yield of labeled proteins and that the binding reaction was 50 times faster than with current methods," Deiters says. "Additionally, it took less reagent to complete the reaction, so overall we have a faster, more efficient method for protein labeling, and less chance of interfering with the normal function of the proteins and cells being studied."
The research was funded by the National Institutes of Health and the National Science Foundation. The Department of Chemistry is part of NC State's College of Physical and Mathematical Sciences.
Note to editors: Abstract of the paper follows
"Genetically encoded norbornene directs site-specific cellular protein labelling via a rapid bioorthogonal reaction"
Authors: Alexander Deiters, Jessica Torres-Kolbus, Chungjung Chou, North Carolina State University; Jason W. Chin, Kathrin Lang, Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, UK
Published: Feb. 5, 2012 in Nature Chemistry
Abstract: The site-specific incorporation of bioorthogonal groups via the expansion of genetic code provides a powerful general strategy for site-specifically labelling proteins with any probe. However, the slow reactivity of the bioorthogonal functional groups that can be encoded genetically limits the utility of this strategy. We demonstrate the genetic encoding of a norbornene amino acid using the pyrrolysyl transfer RNA synthetase/tRNACUA pair in Escherichia coli and mammalian cells. We developed a series of tetrazine-based probes that exhibit 'turn-on' fluorescence on their rapid reaction with norbornenes. We demonstrate that the labelling of an encoded norbornene is specific with respect to the entire soluble E. coli proteome and thousands of times faster than established encodable bioorthogonal reactions. We show explicitly the advantages of this approach over state-of-the-art bioorthogonal reactions for protein labelling in vitro and on mammalian cells, and so demonstrate the first rapid bioorthogonal site-specific labelling of a protein on the mammalian cell surface.
Tracey Peake | EurekAlert!
A landscape of mammalian development
21.02.2019 | Max-Planck-Institut für molekulare Genetik
Atopic dermatitis: elevated salt concentrations in affected skin
21.02.2019 | Technische Universität München
Up to now, OLEDs have been used exclusively as a novel lighting technology for use in luminaires and lamps. However, flexible organic technology can offer much more: as an active lighting surface, it can be combined with a wide variety of materials, not just to modify but to revolutionize the functionality and design of countless existing products. To exemplify this, the Fraunhofer FEP together with the company EMDE development of light GmbH will be presenting hybrid flexible OLEDs integrated into textile designs within the EU-funded project PI-SCALE for the first time at LOPEC (March 19-21, 2019 in Munich, Germany) as examples of some of the many possible applications.
The Fraunhofer FEP, a provider of research and development services in the field of organic electronics, has long been involved in the development of...
For the first time, an international team of scientists based in Regensburg, Germany, has recorded the orbitals of single molecules in different charge states in a novel type of microscopy. The research findings are published under the title “Mapping orbital changes upon electron transfer with tunneling microscopy on insulators” in the prestigious journal “Nature”.
The building blocks of matter surrounding us are atoms and molecules. The properties of that matter, however, are often not set by these building blocks...
Scientists at the University of Konstanz identify fierce competition between the human immune system and bacterial pathogens
Cell biologists from the University of Konstanz shed light on a recent evolutionary process in the human immune system and publish their findings in the...
Laser physicists have taken snapshots of carbon molecules C₆₀ showing how they transform in intense infrared light
When carbon molecules C₆₀ are exposed to an intense infrared light, they change their ball-like structure to a more elongated version. This has now been...
The so-called Abelian sandpile model has been studied by scientists for more than 30 years to better understand a physical phenomenon called self-organized...
11.02.2019 | Event News
30.01.2019 | Event News
16.01.2019 | Event News
21.02.2019 | Life Sciences
21.02.2019 | Earth Sciences
21.02.2019 | Life Sciences