Mutant Chronicles: The quest for a better red-fluorescent protein

Red-fluorescent protein labeling cellular structure in Zebra finch rotundus. The picture is one of many examples of the remarkable insight into cellular structure and dynamics obtained by using fluorescent proteins. The development of new red-fluorescent proteins resistant to inactivation by laser light and oxygen is a goal of the Jimenez group. Credit: Photo courtesy of Dr. Richard Mooney, Duke University School of Medicine
Microfluidics system used in the Jimenez lab to select the top-performing cells during a directed evolution experiment. Lasers measure the fluorescent properties of mutated fluorescent proteins in each cell passing through the system, and another laser provides a “tractor beam” to grab onto the best mutant cells for further investigation. This system allows the group to rapidly investigate tens of thousands of different proteins. Credit: The Jimenez group and Steve Burrows, JILA

Because red fluorescent proteins are important tools for cellular imaging, the Jimenez group is tailoring them to its own biophysics research. The group’s quest for a better red-fluorescent protein began with a computer simulation of a protein called mCherry that fluoresces red light after laser illumination. The simulation identified a floppy (i.e., less stable) portion of the protein “barrel” enclosing the red-light emitting compound, or chromophore. The thought was that when the barrel flopped open, it would allow oxygen in to degrade the chromophore, thus destroying its ability to fluoresce.

The group decided that its next step(s) would be to tweak the natural protein to make it more stable. Tweaking proteins is a huge challenge because most combinations of mutations result in a complete loss of the necessary structure to maintain fluorescence. Even so, the group succeeded in developing a new approach to real-world protein improvement that employs a laboratory strategy for directed evolution.

Directing evolution is challenging. The first step requires creating a library of hundreds of thousands of cells containing different mutations of a single protein. This step is now relatively easy, thanks to the tools of molecular biology. The second step requires screening the fluorescence properties of each cell to select only those few that contain top-performing mutant proteins.

To accomplish the selection process, the group uses microfluidics combined with several laser beams. Its microfluidics system contains micron-sized three-dimensional transparent channels that carry small streams of liquid and allow cells to flow through them one at a time. As the mutant cells pass through the microfluidics channel, lasers measure the fluorescent properties of each mutant cell to assess how well the cells maintain their fluorescence when repeatedly excited by the series of laser beams. Another laser acts as an optical trap that works like a tractor beam to grab onto the best mutant cells for further investigation. The microfluidics setup itself readily removes the cells that are poor performers by simply allowing them flow out of the device.

To make matters more challenging, directed evolution requires repeating the two steps described above multiple times. The Jimenez group is currently in the middle of round three of its quest to evolve a better red-fluorescent protein.

Although the group has already shown that the specific improvements suggested by the computer simulation don’t work, the first round of the directed evolution experiment has come up with an improved red-fluorescent protein with a less floppy barrel that is 2–4 times more stable than mCherry. The combination of mutations that resulted in this improvement has not been previously observed in nature and was completely unexpected.

The group named its new mutant protein Kriek, after a Belgian beer made via the fermentation of cherries. Clearly, the researchers are adept at doing more than biophysics. They include JILA Ph.D. Jennifer Lubbeck (2013) and Fellow Ralph Jimenez, Kevin Dean and Amy Palmer of CU’s Department of Chemistry and Biochemistry, as well as colleagues from the University of Tennessee Space Institute and Florida International University.