On the seventh floor of Kenan Laboratories, afternoon sun beating through the south windows warms the hallway to an uncomfortable temperature. Ice machines hiss and groan, trying to stay cool. On a neglected blackboard nearby is a drawing of a cactus, and a scribbled “Science is kicking my ass right now.”

From around the corner, a faint, whistled rendition of the Indiana Jones theme grows louder. Chemistry professor Gary Pielak strides down the hall in a pair of high-water Levi’s and socks with sandals. Among the prized possessions in his office: a fake raccoon-skin cap he bought on eBay, and a plaque from the National Institutes of Health. He is the first UNC scientist to win an NIH Pioneer Award, which will give him $2.5 million for five more years of protein research. He has worked long and hard for it.

“I’ve been thinking about proteins since Richard Nixon was president, and it just blows me away,” he says. “Still, there are so many things we don’t know.”

Proteins and pandas

Proteins, which are made up of amino acids, have many roles in the cell. For example, some regulate or speed up chemical reactions. Some help a cell stay structured, and others act as channels to control the flow of fluid or ions through membranes. Still others regulate how genes turn on and off. Understanding the spatial structure of proteins helps scientists determine what proteins do and how they do it. Once they know how proteins are supposed to work, scientists such as Pielak can figure out how they’re misshapen in diseases or other conditions.

But take a protein out of its natural environment, the cell, and you may not be able to learn much about it.

It’s kind of like studying a panda in a zoo, says Brian McNulty, who worked with Pielak as a graduate student. You might learn a lot about a panda while it’s in captivity, but you’ll learn more about one while it’s munching bamboo in the mountains of China.

The natural environment of a protein is a cell filled with many proteins, Pielak says. And if he’s been thinking about proteins since the Nixon years, he’s been trying to develop a way to see them in their natural environment since Reagan was president. Not many have tried it. Not many would fund it. And, especially for Pielak’s students, it hasn’t always been easy.

Early in his career, Pielak says, he sat in committee meetings while his students presented their work on protein stability. Back then, a student isolated protein from cells, then measured the protein’s stability while it bathed in dilute buffer solutions. “This is all well and good,” another committee member said to Pielak.“But proteins live in cells, not buffer solutions. And cells are crowded with things.”

“It bugged the heck out of me,” Pielak says. As an assistant professor, he had to crank out reliable experiments, publish, and publish. “When I was finally a tenured professor, I took advantage of what tenured professors are supposed to do — work on something wacko,” he says.

He followed his colleague’s advice and started to work on proteins in their natural environment — living cells. In his case, the living cells were from E. coli.

A sea of proteins

But it’s a challenge to study one protein in a cell, where it’s in a sea of many proteins and other large molecules, Pielak says. Right now, the only way to get atomic-level information about a protein in solution is to lower it into a nuclear magnetic resonance spectrometer (NMR). Carolina’s NMR looks like a six-foot-tall tin can, and Pielak’s group affectionately calls it “the magnet.”

The magnet tells Pielak about the atomic environment of his “favorite” protein. The atomic environment can give clues about whether the protein is folded in certain conditions, which can help determine whether it can carry out its function in the cell, he says.

Before the year 2000, only one other lab had published its findings on NMR inside cells. But that year, Pielak and then-undergraduate Matthew Dedmon began a “swing-for-the-fences experiment,” Pielak says.

Pielak knew of a certain bacterial protein they might detect using in-cell NMR. They knew that it unfolded and lost its structure when in dilute solution, but, when crowded by other proteins, it partially folded and gained some structure. The protein, categorized as intrinsically disordered, was a type that could only be studied in a living cell instead of in isolation, Dedmon says.

So they tried it out both ways — in living cells and alone in the magnet.

Dedmon learned how to use the magnet and worked for about two months on preparing his samples. He remembers carrying his iced E. coli cells to the magnet on many hot summer days, careful to keep them alive during their journey.

One day, it worked. Dedmon processed the data and took the printouts to Pielak — a twenty-minute hike from the magnet to Kenan Laboratories — to show him the fuzzy but readable spectra. It was clear to them that they had something. They could take readings of this protein in a sea of many proteins, in a sea of many E. coli.

They published the findings in 2002.

Playing protein favorites

Pielak’s strategy for students has been “one student, one protein,” he says. Once a student finds a detectable protein, that student can work for years on altering the protein itself or conditions that may affect the protein’s properties. With so many variables to alter, the possibilities for one protein alone are endless.

First, Pielak’s group tells E. coli cells to make a lot of a certain protein in an environment enriched with an isotopic label, 15N. Then they take a sample of the cells — which looks like a straw full of melted vanilla shake — and load it into the NMR spectrometer.

Pielak’s group does things differently from other labs, says graduate student Lisa Charlton.

“We’re sticking live E. coli into an NMR tube, and we have no idea what’s going to happen with each experiment.”

Two-thirds of the proteins don’t “work” in the magnet, and it takes anywhere from two weeks to a month to figure that out, Pielak says. The problem is that the insides of cells are packed with proteins, twice as many as are found in a goopy egg white. So it’s hard to see one protein amid the others. Sometimes a protein is too large. Sometimes it can attach to other molecules or the cell membrane. Or the group can’t get cells to make enough of the protein.

Charlton found her favorite protein about three years ago, and it has been rife with problems ever since.

First, her cells settled into the sensitive spot of the detector region of the magnet. Then she noticed that the cells acidified after a few hours in the magnet. Once, she even had to create twenty variants of the protein. Each problem took her an entire summer to solve, she says.

Trouble in protein paradise

Pielak’s new path had its drawbacks. Though his work is basic science, he knew it could apply to proteins involved in diseases, he says. In his application for the Pioneer Award, he wrote a paragraph called “Persistence in the face of failure.” He had submitted nine applications to NIH over five years, he wrote. None was awarded. His reviewers wrote that the work was “not highly developed enough” or “too novel.” No one wanted to take a chance on his work.

Smaller grants from the National Science Foundation maintained the research in the lab, he says. He had students apply for research scholarships. His lab shared equipment with other labs, which relieved some of the costs of maintenance and repair.

The lab works together, Charlton says. They wash dishes, refill tanks of deionized water, and order materials.

“When something breaks, we don’t call someone in to fix it,” she says. “We fix it ourselves.”

Proteins galore

More cells are on the horizon for Pielak. With the Pioneer funds, he plans to develop equipment to measure the proteins in yeast, insects, and eventually human cells. The lab will use the money to design attachments for keeping these “higher-maintenance” cells alive while they sit in the magnet, he says. Though the money will fund Pielak’s work in the field of in-cell NMR, Charlton says some things will stay the same: “We’ll still be doing our dishes.”