A digital signal processing technique long used by statisticians to analyze data is helping Houston scientists understand the roots of memory and learning, Alzheimer's and Parkinson's diseases and stroke.
Researchers at Rice University and the University of Texas Health Science Center at Houston (UTHealth) reported today in the journal Nature Chemical Biology that single molecule fluorescence resonance energy transfer (FRET) techniques combined with wavelet transforms have given them a new view of the AMPA receptor, a glutamate receptor and a primary mediator of fast signal transmission in the central nervous system.
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," said Christy Landes, a Norman Hackerman-Welch Young Investigator Assistant Professor of Chemistry at Rice and lead author of the new paper. "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 said. 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 said, 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," she said. "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. That led the researchers to employ a unique combination of technologies. Vasanthi Jayaraman, an associate professor in UTHealth's Department of Biochemistry and Molecular Biology 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 Landes and her team 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 said. "In a short period of measurement, we might be counting 10 photons."
The trick, she said, 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 said. "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. (Another recent paper by Landes and Taylor on their wavelet optimization method appears here.)
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 -- say, 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, said 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 said 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 said. "I think our true contribution is to be able to analyze this noisy data to get to what's underneath."
Co-authors of the paper are Anu Rambhadran, a graduate student at UTHealth, and Rice graduate students J. Nick Taylor and Ferandre Salatan.
The American Chemical Society Petroleum Research Fund, the National Institutes of Health and the American Heart Association supported the research.
Read the abstract at http://www.nature.com/nchembio/journal/vaop/ncurrent/abs/nchembio.523.htmlDownload horizontal or vertical illustrations of the receptor here: http://www.media.rice.edu/images/media/NEWSRELS/0203_AMPA_1.jpg
CAPTION: Crystal structures of the GluA2 receptor show the C-clamp-like binding sites and the positions of fluorescent labels (the blue and red ovals) used to measure the sites in the apo (or agonist-free) and glutamate-bound forms. (Credit: Rice University/University of Texas Health Science Center)
Download a high-resolution photo of the research team here: http://www.media.rice.edu/images/media/NEWSRELS/0203_landes.jpg
CAPTION: Researchers at Rice University and the University of Texas Health Science Center at Houston are using a unique combination of techniques to analyze the workings of neurotransmitters. From left, Rice graduate student J. Nick Taylor; UTHealth graduate student Anu Rambhadran; Christy Landes, an assistant professor of chemistry at Rice; and Vasanthi Jayaraman, an associate professor at UTHealth. (Credit: Jeff Fitlow/Rice University)
Located in Houston, Rice University is consistently ranked among the nation's top 20 universities by U.S. News & World Report. A Tier One research university known for its "unconventional wisdom," Rice has schools of Architecture, Business, Continuing Studies, Engineering, Humanities, Music, Natural Sciences and Social Sciences and offers its 3,485 undergraduates and 2,275 graduate students a wide range of majors. Rice has the sixth-largest endowment per student among American private research universities and is rated No. 4 for "best value" among private universities by Kiplinger's Personal Finance. Its undergraduate student-to-faculty ratio is less than 6-to-1. With a residential college system that builds close-knit and diverse communities and collaborative culture, Rice has been ranked No. 1 for best quality of life multiple times by the Princeton Review.
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