Valerica Raicu's lab is capturing the promise of better drugs through imaging protein interactions in living cells.
Proteins are sequences of amino acids whose three-dimensional shape determines their function in living cells. They are the main components of cellular material, serving as enzymes, hormones, structural elements, receptors, and antibodies. A common estimate is that there are over 100,000 unique types of proteins in the human body.
In the pharmaceutical industry, cell membrane proteins are particularly important. They control the flow of information and materials between cells and mediate activities such as nerve impulses and hormone action. Many diseases involve disruption of the essential processes of membrane proteins, and some 60 percent of drugs target them.
An important discovery by scientists in recent years is that multiple proteins usually work together to perform cellular tasks.
"Proteins rarely do something of biological significance alone," explains Valerica Raicu, an assistant professor of physics at UWM whose research focuses on membrane proteins. "They usually aggregate and form small nanomachines that have a biological role, accomplish a biological function."
Raicu is among the growing number of scientists interested in the physics of biological processes. "Our main goal here is to visualize proteins inside the living cell," he says, standing beside the laser-scanning microscope in his laboratory. "And not only to visualize them, but to detect any possible interactions they may have with other proteins, of their own type or a different type."
As the Human Genome Project sequenced and mapped the entire collection of DNA in human beings, scientists learned the function of genes and their location on the chromosome. "The next step is to understand how the proteins, which are actually the product of genes, function," Raicu explains.
The proteins Raicu is studying are a family of transmembrane receptors known as G protein-coupled receptors (GPCRs), which mediate cellular responses to signaling molecules such as hormones and neurotransmitters, as well as local mediators including proteins, small peptides, amino acids, and fatty acid derivatives.
To capture images of cells, which are often measured in micrometers, or microns—hundred-thousandths of a meter—Raicu's team built its own laser-scanning microscope. "Because of the types of questions we ask, we need new technology," he says. "There is no technology available to answer our questions."
The team needed a microscope that could scan the cell rapidly and in very thin sections. With its tightly focused laser, the microscope can capture slices as thin as 300-400 nanometers, or billionths of meters. The cell Raicu uses as a model—yeast—is about 5-6 microns. "But most cells are larger than that, so really you can get very nice high-resolution images," Raicu says.
Thanks to the microscope's CCD camera—its image sensor features an integrated circuit with an array of linked light-sensitive capacitors—and an ultrashort pulse laser, whose pulses last just 10 femtoseconds (that equals one hundred trillionths of a second, or 0.00000000000001 second), the microscope can capture about 150 frames per second. "Most of this speed is spent on gaining color information of the visualized sample, but enough remains available for fast imaging of the cell," Raicu says.
Even with high-resolution, high-speed movies, Raicu still has to know what he's looking at. To help with that, members of Raicu's team travel to the lab of Chaoyang Zeng, a UWM assistant professor of biological sciences, to genetically modify their GPCR proteins. With help from Zeng, as well as from David Jansma, a collaborator at the University of Toronto in Canada, they have synthesized the proteins with fluorescent tags—molecules that emit light when excited by the laser. They use the green fluorescent protein (which comes from the jellyfish Aequorea Victoria) and differently colored variants to tag the different proteins they're interested in. This way Raicu can track the positions and movements of the proteins under his laser-scanning microscope.
To detect interactions between proteins, Raicu takes advantage of a special exchange of energy—Förster resonance energy transfer (FRET)—that occurs when two fluorescent molecules come within a nanometer of each other. During FRET, a laser-excited molecule, referred to as the donor, transfers its energy to a nearby molecule, called the acceptor, and only the acceptor emits light. So if the donor normally fluoresces green, but only yellow is emitted (by the acceptor), Raicu knows that FRET has occurred, and that the two membrane proteins have interacted.
"We can detect whether or not they are interacting, whether they're close to each other, if they exchange energy, and then see a shift in the color," he says "We do a lot of mathematics to quantify that, find out how many are interacting, how many are not interacting, and so on."
When fully developed, Raicu's technology should be an important tool in drug development.
"People working in the pharmaceutical industry will be interested to tailor their drugs to the properties of their proteins," he says. If those receptors are distributed uniformly on the membrane, that's an important question. If they are forming complexes, getting together, that's important. If they do, what's the size of those complexes?
"Then, knowing that, you know what kind of drug to design. Those guys would need this kind of toy for drug design."
Raicu estimates his technology could be integrated into a commercial microscope and available to medical and pharmaceutical professionals within five years. ♦