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Shining a Light on Fluorescent Proteins

Sarah C.P. Williams

By comparing the structures of different cyan fluorescent proteins, researchers figure out what makes some brighter than others—and how to make one even brighter. 

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When structural biologist David von Stetten set out to look at the 3-D arrangement of fluorescent molecules, his goal was to answer a basic scientific question: how do structural elements affect a molecule’s brightness? But in the end, von Stetten discovered something else—how to make an even brighter cyan protein.

After scientists had begun attaching green fluorescent protein (GFP) to proteins to visualize the molecules during the 1990s, they then developed different colored versions of the protein, like yellow (YFP) and cyan (CFP), to label multiple targets within a single experiment.

Crystals of mTurquoise2, like the one shown above, let researchers study what makes some fluorescent molecules shine more brightly than others. Credit: von Stetten/Royant/CNRS-ESRF

“The problem with these variants was that they were all artificial, and hadn’t gone through any rounds of natural evolution,” says von Stetten of the European Synchrotron Radiation Facility in France. “They were altered to change the color they emitted, but this also changed the structure of the whole protein and made it weaker.”

So, scientists made educated guesses as to what further mutations could make these variants brighter, leading to versions with improved quantum yields, like enhanced cyan fluorescent protein (ECFP) and super cyan fluorescent protein 3A (SCFP3A). If a protein’s quantum yield is 100%, it emits all the light shone on it as fluorescence. Early versions of cyan proteins, however, had quantum yields around 36%; the latest version—mTurquoise—has a quantum yield of 83%. To understand how the mTurquoise’s structure improved its quantum yield, von Stetten and colleagues crystallized different proteins in the cyan family and looked at the arrangement of amino acids within them.

“We found some explanation why mTurquoise is brighter,” says von Stetten. “And it mainly has to do with the overall stability of the protein’s structure.” In a less-bright cyan fluorescent protein, floppy amino acids hit against the chromophore—the crucial part of the protein responsible for its color—and destroy its fluorescence. The brighter mTurquoise had mutations that led to fewer floppy bits near the chromophore.

“What surprised us what how little changes have such big effects,” says von Stetten. “Basically a single change in a hydrogen bond can change the quantum yield from 36–56%.” As they analyzed mTurquoise’s structure in further detail, they soon realized that there was still one weak spot.

So, von Stetten’s collaborators, a team of biologists at the University of Amsterdam, replaced the weak amino acid. While 18 of the 19 possible replacement amino acids decreased mTurquoise’s brightness, one improved it. They dubbed the new protein mTurquoise2. Not only was it brighter than mTurquoise, with a quantum yield of 93%, but it also performed better in other lab testing, shining for a longer time (1).

“The science that’s done with these kinds of proteins is always at the detection limit,” says von Stetten. “So, by increasing that detection limit by even 10%, it opens up a whole new level of experimentation that wasn’t possible before.”

Next, von Stetten’s team plans to tackle other fluorescent molecules, such as the popular yellow variants, to determine if there are ways to further stabilize their structures. “There’s a whole rainbow of colors that exists now and so we think there’s probably room for improvement in some of the other fluorescent proteins as well,” he says.


1. Goedhart, J., D. von Stetten, M. Noirclerc-Savoye, M. Lelimousin, L. Joosen L, M.A. Hink, L. van Weeren, T.W.J. Gedella, and A. Royant. 2012. Structure-guided evolution of cyan fluorescent proteins toward a quantum yield of 93%. Nature Communications 3:751.

Keywords:  protein