The use of fluorescent proteins such as GFP has been a mainstay of biological analysis for decades and has even garnered the discoverers a Nobel Prize. While these molecules are well suited for laboratory use, they do have their limitations, as they are vulnerable to heat, can be bulky in protein structure, and only glow in the presence of oxygen. However now, biophysicists from the Moscow Institute of Physics and Technology have joined forces with colleagues from France and Germany to create a new fluorescent protein. In addition to glowing when irradiated with ultraviolet and blue light, the new molecule is exceedingly small and stable under high temperatures.

Findings from the new study—published recently in Photochemical & Photobiological Sciences through an article titled, “A thermostable flavin-based fluorescent protein from Chloroflexus aggregans: a framework for ultra-high resolution structural studies”—shows that the protein holds renewed prospects for fluorescence microscopy.

Fluorescence microscopy is a method for studying living tissue that relies on induced luminescence. After being exposed to laser radiation at a particular wavelength, some proteins emit light at a different wavelength. This induced “glow” can be analyzed using a special microscope. Researchers append such fluorescent proteins to other proteins via genetic engineering to make the latter ones visible to the microscope and observe their behavior in cells. Moreover, extended exposure heats up the fluorescent proteins like GFP, shortening their visible lifespan.

3D structure of the new fluorescent protein. [Vera Nazarenko et al.]
“Our protein is more thermostable than its analogs: It only denaturates at 68 degrees Celsius,” said the paper’s lead author Vera Nazarenko from the MIPT Laboratory of Structural Analysis and Engineering of Membrane Systems. “It is also miniature, while most of the currently used fluorescent proteins are rather bulky. On top of that, it can emit light in the absence of oxygen.”

The research team originally identified the protein with these remarkable properties in the cells of a thermophilic bacterium—that is, one which lives in high-temperature environments, such as hot springs. The researchers then genetically engineered a DNA sequence that reproduced the protein’s fluorescent segment but not the other parts, which would make the molecule larger.

By introducing the gene that encodes the protein into the cells of another bacterium, Escherichia coli, the team turned it into a factory mass-producing the fluorescent protein with unique properties.

“We present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans,” the authors wrote. “CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68°C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small-angle scattering data show that the protein is a dimer.”

Researchers studying the processes that occur in living cells have been waiting for a protein combining these crucial features for a long time. By introducing it into cells, they can now obtain essential data on cell life and death. To name a few applications, fluorescence microscopy is seen as one of the best tools for investigating the mechanism behind malignant tumor genesis and development. It is also useful for research on cell signaling and organ development.

“Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter,” the authors concluded.

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