New study shines light on how to better engineer fluorescent proteins

Researchers have now captured the ultrafast changes of green fluorescent proteins as they transition between a dark and fluorescent state, using an X-ray laser at the SLAC National Accelerator Laboratory.

Green fluorescent proteins (GFPs), originally found in the jellyfish Aequorea victoria, have helped transform biomedical research. Their green glow has acted like a flashlight on the inner workings of cells, illuminating pathways and processes in lab dishes and living animals since it was discovered in 1961. The protein acts as a molecular switch depending on the conditions, flipping from dark to glowing when excited by light. Scientists attach these fluorescent tags to other proteins to track their activity — studying how cancer cells spread, how HIV infections progress, how genes are expressed and much more.

Although researchers have used these proteins for decades, they were unable to observe how GFPs flipped between their dark and glowing states until now. The transition was too fast for traditional X-ray imaging techniques. So an international collaboration of scientists recently used SLAC’s Linac Coherent Light Source, one of the world’s fastest and brightest X-ray lasers, to excite the proteins and take snapshots of the fluorescent molecules in action.

These images were used to investigate what happened as GFP flipped states — with the hope of engineering GFP to make this happen even faster. They found that the protein became momentarily stuck between a dark and glowing state, as reported in Nature Chemistry.

“After a picosecond, a very short time, this molecular switch is stuck between on and off,” said Martin Weik, PhD, a scientist at the Institute of Structural Biology in Grenoble, France, in a recent news release. “People have predicted this, but to actually confirm it experimentally is extremely exciting. It’s as if there is a door and it’s neither closed nor completely open; it’s half open. And now we are learning what can go through the door, what might be blocking it and how it works in real-time.”

The team discovered that an amino acid partially blocked the doorway, slowing the GFP’s ability to flip states. Using this knowledge, they then engineered a mutated version of the protein with a smaller amino acid that could switch more quickly — creating a brighter and more efficient fluorescent tag that can observe cellular processes more precisely.

“We think that this approach will open a world of possibilities to tailor and create proteins,” Weik said in the release. “We not only have the structure of the molecule, but now we can see what is happening between one static state and the other.”

This is a reposting of my Scope blog story, courtesy of Stanford School of Medicine.

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