A Passion for Precision
Biological structures do not like to be measured. But this does not stop Gavin King.
avin king’s laboratory is deep down in the basement of MU’s hulking Physics and Astronomy Building, not by choice but necessity.
The slightest vibration, whether it’s traffic outside or people hurrying down a hall, can upset his work. King is using a unique $300,000 microscope, largely of his own design, to create three-dimensional images of cell membranes down to just a couple of atoms.
But its subterranean location alone isn’t enough to isolate this atomic force microscope. Inside his lab, King has built a separate isolation room, its walls inside and out covered with panels of acoustical foam. The table on which the microscope rests floats on a cushion of air. No one is allowed in the room while the microscope is being used. Even everyday sounds or temperature changes can disturb this sensitive instrument’s measurements.
“One thing that motivates me is performing precise measures of biological structures,” says King, who holds appointments at the University of Missouri in both physics and biochemistry.
“Biology is squishy and measurement is hard,” he says. Apply a set of calipers to your finger; you can get different readings of its diameter, depending on how firmly you press. “Biological structures don’t like to be measured.”
Gavin King is using his atomic-force microscope to explore how individual protein molecules behave on the surface of cells they inhabit.
King’s field is called single molecule biophysics. He is exploring how individual protein molecules behave on the surface of the cells they inhabit.
That’s important to know because the proteins serve as cells’gatekeepers, creating channels that let substances pass in and out through the membranes that hold cells together.
More than half of all current and future drugs target membrane proteins. A better understanding of how these proteins work may prove invaluable to the development of drugs that can be better absorbed by particular cells.
King’s methods are a far cry from usual laboratory techniques that analyze large collections of proteins and average out their activity. Such “bulk biological techniques,” as King calls them, are like watching a foot race from a balloon overhead. “You can see the general flow of what’s happening, but not what individual racers are doing.”
But to see these individual proteins takes technology that drills down to the “nano” level. A nanometer is one billionth of a meter. To begin to comprehend just how small that is, consider that a human hair is 50,000 nanometers in diameter. Cell membranes are just four nanometers thick.
The atomic force microscope is the culmination of a century of technology — x-ray diffraction, field ion microscopy, scanning tunneling microscopy — making it possible for scientists to visualize what happens at the atomic level. “I like to frame this in terms of man’s quest to see an atom,” King says. “In principle, we can see atoms with this.”
Granted, with the squishiness of biology, the best resolution of membrane proteins that King achieves is about two or three atoms. Still, that’s a mere fraction of a nanometer.
The gold standard technique for studying the structure of proteins has been x-ray crystallography. But this technique removes proteins from their biological environment. With the atomic force microscope, King’s laboratory can keep the proteins in room-temperature fluid, similar to what’s found in cell membranes.
King says there’s evidence now that the crystallized versions of proteins need for x-ray crystallography are significantly different from the active versions seen under his microscope. “Atomic force microscopy will never trump x-ray crystallography, but we can add more truth telling,” he says.
The atomic force microscope was invented in the mid-1980s by physicists in Zurich and at Stanford University to look for defects on the surfaces of silicon compounds, such as gallium arsenide, used in computer chips.
“They were basically surface scientists, looking at solid state surfaces,” King says. “Then others realized this tool has a really bright future in biology.”
For all its sophisticated technology, the atomic force microscope uses something that looks a lot like a phonograph needle, a tip just a few nanometers long, attached to a short horizontal cantilever.
The tip of the cantilever, made out of silicon like a microchip, is run across the surface of whatever is being studied, causing the cantilever to flex up and down. A laser beam bounced off the cantilever measures its movement. The needle itself remains stationery as the sample is moved back and forth and longitudinally to scan its surface. King compares the way the microscope works to reading Braille.
“The whole instrument is based on touch; it’s a touchy-feely microscope,” King says. “It’s a powerful tool, as long as you have a sharp tip.”
The microscope has additional technology to keep the needle’s tip from digging into the sample, which can put a trough in the cell membrane and contaminate the needle. Even so, biological molecules can be sticky.
“We like to work with the sharpest tips we can, but we go through a lot of tips,” says King. Luckily, the needles cost as little as $20.
King’s atomic force microscope is a souped up version of the original. It has two extra lasers and parts fabricated at MU’s physics machine shop to create three-dimensional images. King has received grants from the National Science Foundation and the Burroughs Wellcome Fund for his work.
While conventional atomic force microscopes measure the up and down motion of the sensor tips, King’s device also measures the side-to-side twists of the tip providing the 3-D images. King has also improved on the microscope’s precision to account for the inevitable shifting of biological samples. In the past, scientists kept their atomic-force microscope at ultra-low temperatures in order to slow the movement of the molecules they were studying and to stabilize the measurements. But that’s not a natural environment for biologically active proteins.
Scientists have tried to get around the difficulty of “thermal drift” at higher temperatures by taking a series of quick snapshots of a molecule. But the molecule’s ever-shifting position makes for less-than-satisfactory results.
King solves the problem by using a laser to track the position of the sample, just as another laser tracks the movement of the needle’s cantilever. Coordinating these two measures allows the microscope to stabilize the position of the needle’s tip with respect to the sample.
“This is the only instrument of its kind in the world,” King says of his ultra stable atomic force microscope. “We have a force microscope with bells and whistles.”
King is collaborating with MU biochemist Linda Randall, as well as scientists at Johns Hopkins and the University of California-Irvine. In his lab, King works with postdoctoral scholar Krishna Sigdel and a team of graduate and undergraduate students.
King and Sigdel want to know what the mosaic of proteins and lipids that make up cell membranes look like, and how they change, for example, when a drug is present.
The membranes serve as a container for the cell’s cytoplasm, giving the cell shape and connecting it to other cells. They are made up of lipids — fats, in layman’s terms — that assemble themselves into double layers. “You can think of it as an oil drop that envelops the cell,” says King.
Various proteins insert themselves into the membranes to form the mosaic structure; these surface proteins account for more than 30 percent of the proteins that most cells express. Cell membranes naturally tend to repel water. The proteins serve as conduits that allow essential nutrients, such as amino acids and sugars, to enter the cell. The images King has gotten show these proteins like stars in a night sky. The microscope can measure how high the proteins rise above the membrane surface. Over time, some pop up or change their shape or position.
“Is this interesting biologically? We think the answer is ‘yes,’” King says.
On King’s to-do list is observing proteins being pushed out through this complex of proteins. This will be a step toward eventually showing how the proteins change when they bind with a drug.
One technique he is using is to put a protein on the microscope’s needle tip, dip it down to the cell membrane and then pull it up again to see how the experimental protein interacts with the proteins on the cell’s surface.
“We can hover the tip wherever we want for a very long time on the molecular scale. We could potentially send in a drug and then see how it binds.”
His lab is doing that now with melittin, a powerful toxin in bee venom that can rip holes in membranes. Melittin is so potent, other researchers are studying its potential as a cancer drug and as an anti-HIV agent.
King is taking requests for microscope time from other scientists at MU and expects to make it available to researchers on other campuses.
But he’s also reaching out beyond the scientific community. He believes it’s important that scientists stay in touch with the public.
King has collaborated with MU faculty from the physics, education and biochemistry departments on a week-long summer camp program, “Biophysics and Your Body,” for middle school students. The camp’s curriculum has been turned into a series of five lesson plans — on ears, eyes, muscles, energy and metabolism — that teachers can incorporate into their classes.
King also co-chaired an MU program that offers graduate level instruction to scientists on how to better engage the public about the research they do.
Scientists can often be aloof, King says, and no matter how good their work is, that kind of attitude does a disservice to science in general.
“The scientific effort as a whole requires investment from people who are not scientists. The people who keep the lights on in my lab, that are funding our research, are the average people that are paying their taxes,” King says.
“Unless we as a scientific body communicate effectively to the general public, I think we have a real problem on our hands in terms of justifying to the taxpayers supporting what we do.”
While his mop of blond hair and his informal manner may lead you to assume he had a Midwestern, Tom Sawyer childhood, King is actually a New York City guy, raised on the Upper East Side of Manhattan. It wasn’t until he was well into his career that he moved to the Heartland.
Another incorrect assumption that would be easy to make is that he must have been a science geek as a child. King was always good at math and science at school but he never had a passion for them. In high school, he found conventional Newtonian physics boring. “As a kid, I liked to take things apart, which could get me in trouble if they belonged to my older brother.” He also built model cars and airplanes, most of which didn’t work.
King went off to Bates College in Maine assuming he probably would end up as a political science or history major. That all changed during his freshman year. King took an introductory course on Einstein’s theories, learning how, for example, time slows as velocity increases. “That just blew my thoughts up. It’s what baited the hook,” he says. King started taking all the physics courses he could and graduated in 1997 with a bachelor’s degree in physics.
From Bates, King went on to Dartmouth College, where he first encountered an atomic force microscope. In it King found the perfect device, one that allowed him to combine his love of physics with his childhood interest in tinkering and building.
The microscope at Dartmouth was built into its own refrigerator for conducting solid-state physics experiments that are done best at ultra-low temperatures. It was here that King started working on ways to extend this kind of precision to biologically relevant conditions and developed technology to cancel out distortions when measuring biological molecules.
At Dartmouth, King also met his wife-to-be, who was working on her doctorate; Karen King now serves as an assistant teaching professor in the department of physics and astronomy at MU. The Kings have a son and daughter: five-year-old Marshall and three-year-old Annabelle.
King received a master’s degree in physics at Dartmouth before moving on to Harvard. There he earned his doctorate in physics while helping to develop technology for low-cost DNA sequencing.
For five years, King was a post-doctoral research associate at JILA — originally called the Joint Institute for Laboratory Astrophysics — at the University of Colorado in Boulder, one of the leading research centers in the physical sciences. With his mentor, Thomas Perkins, he continued his work on atomic force microscopes, finding ways to stabilize their platforms for studying single molecules. The prototype for the microscope he built at MU is still in use in Colorado.
King came to MU in October 2009. A major attraction for King was the university’s full-time machine shop that was able to fabricate many of the parts for his microscope. “It’s just a fantastic place to do research because you have this resource right down the hallway,” he says.
But not everything in King’s microscope has been machined with such precision. Daubed around some of the microscope’s wiring is Silly Putty. It’s an excellent material, King says, for dampening those pesky vibrations.