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"Unnatural" chemical allows researchers to watch protein action in brain cells

Cell

Photo of neurons differentiated from neural stem cells HCN-A94 with an unnatural amino acid incorporated.
Photo: Bin Shen.


Researchers in the lab of Lei Wang have genetically incorporated "unnatural" amino acids, such as those emitting green fluorescence, into neural stem cells, which then differentiate into brain neurons with the luminescent "tag" intact. This work, which appeared in Stem Cells, may help scientists probe the mysteries of many different kinds of stem cells in humans, as well as the cells they produce, and could be a boon to basic and clinical research, helping to speed development of stem cell-based therapies.

Stem cells hold great potential for the treatment of various diseases, yet it has been hard to study how they self-renew and produce all of the body's cells. Incorporating unnatural amino acids will allow researchers to study in real time a particular protein in a living cell or organism, compared to the traditional biochemical methods, which are conducted through such means as a test tube.

Wang and his colleagues pioneered the use of unnatural amino acids (Uaas), which were first incorporated into bacteria in 2001 and mammalian cells in 2007. This latest study, which was conducted in two stages, represented the first use of Uaas in stem cells. In the initial set of experiments, the researchers found that Uaas were successfully incorporated into neural stem cells, the incorporation lasted through the differentiation, and these cells then produced neurons carrying the fluorescent amino acid. The second set of experiments demonstrated how these Uaas can be used to help solve a biological question—specifically how voltage-sensitive ion channels, which are pore-forming proteins, work in neurons.

"We detected changes in fluorescence intensity of the Uaa when the neurons were stimulated," explains Wang, "and these changes are dependent on where the Uaa was incorporated, which hint that different positions of the protein are moving into or outside of the membrane in response to the electric field."

Wang says this experiment can also be adapted to study other membrane proteins in other cells, no matter where they exist in the body.

In related work, reported in Angewandte Chemie, researchers in Wang's lab reported a method for demonstrating how Uaas can be used to map the structure of a corticotropin-releasing hormone receptor (CRF-R1), which regulates human stress. They also show how this new tool helped locate three areas on the receptor to which peptide hormones can dock to activate or inhibit the receptor.