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!
Quality control in immune communication: Chaperones detect immature signaling molecules in the immune system
20.09.2019 | Technische Universität München
Moderately Common Plants Show Highest Relative Losses
20.09.2019 | Universität Rostock
How long the battery of your phone or computer lasts depends on how many lithium ions can be stored in the battery's negative electrode material. If the battery runs out of these ions, it can't generate an electrical current to run a device and ultimately fails.
Materials with a higher lithium ion storage capacity are either too heavy or the wrong shape to replace graphite, the electrode material currently used in...
To process information, photons must interact. However, these tiny packets of light want nothing to do with each other, each passing by without altering the...
Researchers from the Department of Atomically Resolved Dynamics of the Max Planck Institute for the Structure and Dynamics of Matter (MPSD) at the Center for Free-Electron Laser Science in Hamburg, the University of Hamburg and the European Molecular Biology Laboratory (EMBL) outstation in the city have developed a new method to watch biomolecules at work. This method dramatically simplifies starting enzymatic reactions by mixing a cocktail of small amounts of liquids with protein crystals. Determination of the protein structures at different times after mixing can be assembled into a time-lapse sequence that shows the molecular foundations of biology.
The functions of biomolecules are determined by their motions and structural changes. Yet it is a formidable challenge to understand these dynamic motions.
At the International Symposium on Automotive Lighting 2019 (ISAL) in Darmstadt from September 23 to 25, 2019, the Fraunhofer Institute for Organic Electronics, Electron Beam and Plasma Technology FEP, a provider of research and development services in the field of organic electronics, will present OLED light strips of any length with additional functionalities for the first time at booth no. 37.
Almost everyone is familiar with light strips for interior design. LED strips are available by the metre in DIY stores around the corner and are just as often...
Later during this century, around 2060, a paradigm shift in global energy consumption is expected: we will spend more energy for cooling than for heating....
19.09.2019 | Event News
10.09.2019 | Event News
04.09.2019 | Event News
20.09.2019 | Life Sciences
20.09.2019 | Life Sciences
20.09.2019 | Life Sciences