A new study shows which researchers have effectively quenched a debate over the mechanism behind a fluorescent biosensor that monitors neurons by sensing changes in voltage. The study published in the Journal of the American Chemical Society.
The protein is tethered to the neuron's cell wall by a voltage-sensing component that moves a few angstroms when a signal from another neuron changes the electrical charge in the membrane. The Rice researchers theorized that motion pulls the protein against the membrane, compressing it and quenching fluorescence.
A mechanical process controls the quenching of fluorescence in ArcLight, a synthetic voltage indicator placed within proteins that line the inner membranes of neurons. Through their models, the researchers coupled both the mechanism and fluorescence to the strength of electric fields they observed across the chromophore, the fluorescing part of the protein.
Their results showed a simple measure of the field in a simulation could be used to predict whether and how well new fluorescent sensors will behave before researchers synthesize them, Rossky said. ArcLight, developed by Yale neuroscientist Vincent Pieribone in 2012, is a genetically encoded fluorescence voltage indicator protein.
It contains a mutation that makes the fluorescence signal dim when voltage rises and brighten when voltage falls. That makes it useful for tracking signals in the nervous system by expressing the protein in neurons and seeing how they light up. Rossky said changing the shape of the protein brings two residues a nanometer closer to each other.
That's enough to dictate how the chromophore gets rid of energy, either as light as heat. She said a decade long debate between scientists failed to determine whether mechanical or electrical properties of proteins caused their fluorescence. It turned out to be a bit of both.
"A recent paper gave computational evidence for it being predominantly electrostatic, and it kind of makes sense because the protein's very soft," Simine said. "We also figured those mutations are sticking to the membrane, and when they do, the protein's orientation allows the protein to be compressed."
"We can take any mutation of the sequence of this protein and translate it into two numbers that are the inputs for this model, the electrostatic fields around the chromophore. It's a nice, elegant phenomenological theory."
The lab plans to test its technique on custom-synthesized fluorescent proteins and matching simulations to see if their theory and experimentation continue to align. If they do, they expect their models will be highly useful to synthetic biologists making new classes of fluorescent markers.
Author concludes that to understand the fluorescence from a given molecule, they need to conduct experiment and know about the mechanism and the calculations are incredibly valuable