Protein clamps down on neuro diseases

RICE (US) — A digital signal processing technique commonly used to analyze statistical data is clarifying the roots of memory and learning, Alzheimer’s and Parkinson’s diseases, and stroke.

The technique combines single molecule fluorescence resonance energy transfer (FRET) and wavelet transforms to give a new view of the AMPA receptor, a glutamate receptor that is a primary mediator of fast signal transmission in the central nervous system.

Details are reported in the journal Nature Chemical Biology.

Scientists have long thought these receptor proteins, which bind to glutamate to activate the flow of ions through the nervous system, are more than simple “on-off” switches.

A “cleft” in the AMPA protein that looks and acts like a C-clamp and that binds the neurotransmitter glutamate may, in reality, serve functions at positions between fully open (off) and fully closed (on).

“In the old days, the binding was thought to be like a Venus flytrap,” says Christy Landes, assistant professor of chemistry at Rice University.

“The trap sat there waiting for something to come into the cleft. A neurotransmitter would come in and—oops!—it snapped shut on the molecule it was binding to, the gate opened up and ions would flow.

“We have all sorts of high-quality X-ray crystallography studies to show us what the snapped-open and snapped-shut cleft looks like.”

But X-ray images likely show the protein in its most stable—not necessarily its most active—conformation, she says. Spectroscopy also has its limits: If half the proteins in an assay are open and half are shut, the measured average is 50 percent, a useless representation of what’s really going on.

The truth, Landes says, is that the clefts of AMPA receptors are constantly opening and closing, exploring their space for neurotransmitters.

“We know these proteins are super dynamic whether glutamate is present or not. And we need to look at one protein at a time to avoid averaging.”

But seeing single protein molecules go through the motions is well beyond the capability of standard optical tools, leading researchers to employ a unique combination of technologies.

Vasanthi Jayaraman, associate professor of biochemistry and molecular biology at the University of Texas, who studies chemical signaling, started the process when she used the binding domain of the AMPA receptor and attached fluorescent dyes to the points of the cleft in a way that would not affect their natural function.

Single-molecule FRET allowed researchers to detect the photons emitted by the dyes. “These experiments had to be done in a box inside a box inside a box in a dark room,” she says. “In a short period of measurement, we might be counting 10 photons,” Landes says.

The trick was to excite only one dye, which would in turn activate the other. “The amount of light that comes out of the dyes has a direct relationship to the distance between the dyes,” Landes says. “You excite one, you measure both, and the relative amount of light that comes out of the one you’re not exciting depends on how close they are.”

Detecting very small changes in the distance between the two points over a period of time required calculations involving wavelets, a tool Rice mathematicians helped develop in the ’70s and ’80s.

Wavelets allowed the researchers to increase the resolution of FRET results by reducing shot noise—distortion at a particular frequency—from the data. It also allowed them to limit measurements to a distinct time span, for example, 100 milliseconds, during which the AMPA receptor would explore a range of conformations.

They identified four distinct conformations in an AMPA receptor bound to a GluA2 agonist (which triggers the receptor response). Other experiments that involved agonist-free AMPA or AMPA bound to mutated glutamate showed an even floppier receptor.

Knowing how cleft positions match up with the function is valuable, says Jayaraman, who hopes to extend the technique to other signaling proteins with the ultimate goal of designing drugs to manipulate proteins implicated in neurological diseases.

“It was a beautiful combination,” she says of the experiments. “We had done a lot of work on this protein and figured out the basic things. What was lacking was this one critical aspect. Being able to collaborate with a physical chemist (Landes) who had the tools allowed us to get details about this protein we wouldn’t have seen otherwise.”

“Physical chemistry, for all of its existence, amounts to coming up with new tricks to be able to calculate things that nature would not have us calculate,” Landes says. “I think our true contribution is to be able to analyze this noisy data to get to what’s underneath.”

The American Chemical Society Petroleum Research Fund, the National Institutes of Health and the American Heart Association supported the research.

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