Alumnus, faculty achieve first look at biochemical process

Dr. Dennis Stuehr knew he had received an unusually strong undergraduate education in chemistry at BGSU when he got to graduate school at the Massachusetts Institute of Technology (MIT) and found himself more than prepared. Almost 35 years later, his faith in his alma mater was reconfirmed when he found at Bowling Green just the expert he needed to continue his research into the critical biological process of nitric oxide (NO) production.

Dr. H. Peter Lu, Ohio Eminent Scholar in photochemical sciences, is the BGSU faculty member with the rare combination of knowledge, expertise and technical equipment that Stuehr, a researcher with the Cleveland Clinic, was seeking. Their partnership is already yielding rich results of medical and biological importance.

The two have a four-year grant from the National Institutes of Health (NIH), and have just published the first paper on their research, in the leading journal Proceedings of the National Academy of Science (PNAS). In accepting the paper for publication, the journal editor, a Nobel laureate, gave it high praise, said. “He gave very good comments. We were very pleased,” Lu said.

Stuehr, Lu and their team show for the first time precisely how the important calcium-binding protein calmodulin facilitates the production of NO by the NO synthases, which are enzymes that help control innumerable biological processes, including blood pressure, memory and immune defense. The article is titled “Single Molecule Spectroscopy Reveals How Calmodulin Activates NO Synthase by Controlling Its Conformational Fluctuation Dynamics.”

Stuehr, a 1980 BGSU graduate, is an enzymologist studying the means by which the body manufactures NO. “It’s a signal molecule,” Stuehr explained. “People didn’t know it was used in biology until relatively recently, and when that was discovered, in 1987, it led rather quickly to the 1998 Nobel Prize in medicine.”

NO is produced by the body and is the molecule that all animals and humans use to open their blood vessels. “There was an avalanche of interest in the pharmacology community when its vascular function was discovered, and a massive explosion of papers about nitric oxide,” Stuehr said.

In the intervening years, NO has been implicated in functions from gastric motility to immune response to behavior. It has contributed to the development of drugs ranging from blood pressure medications to Viagra.

But how does the body produce it, and how does it work at a molecular level? Those questions have formed Stuehr’s life work and are a source of endless fascination for him. His lab has made numerous discoveries over the years, and as more is known, his interest has only grown.

When he applied for a grant from the NIH to study calmodulin’s role in nitric oxide synthase, the review committee recommended that he collaborate with a single molecule specialist, and that is when Stuehr found Lu at his alma mater.

An elected Fellow of the American Physical Society, Lu has made significant advances in high-resolution microscopy that allows single enzyme molecules to be studied as they undergo structural changes while catalyzing chemical reactions. His work has been recognized internationally. In addition to his studies of single-molecule enzymology and protein dynamics, he conducts studies on a wide range of topics, including solar energy production.

For Stuehr’s calmodulin study, Lu and BGSU postdoctoral fellow researcher Yufan He developed a technique called single molecule photon stamping spectroscopy to elucidate the way in which calmodulin promotes the chemical reaction that creates the NO synthase enzymes by probing the protein conformational fluctuations in the catalytic reaction of producing the nitric oxide molecule.

“He’s gazing at the behavior of single molecules over 60 seconds, which is a long time in enzymatic time frames,” Stuehr said. “There’s been a story building about what calmodulin was doing, but we had to look at it at the molecular level. We had used experimental ways to understand it, from looking at the proteins in solution spectroscopy, but with the single-molecule approach we could finally see what was happening.”

“Each enzyme is almost like an individual person,” Lu said. “They are so complex, and each is unique. The traditional way of studying them, over the last 100 years, has been to average their movements, which are stochastic, or random and not synchronized. But since each molecule is so complex, one simple change in calculation could completely make your results invalid. So we have to have a way to follow one single molecule very precisely and make a ‘movie’ of it so we can know exactly what it is doing.”

Lu compared the process to trying to learn about jellyfish by taking snapshots and calculating on average the movements of a group of them as they open and close, versus filming a single individual in motion to learn how it does what it does. He also offered the historical example of the first time a running horse was captured on film, which proved that all four feet are momentarily off the ground at once — a groundbreaking leap in understanding its locomotion.

Like a person performing a complex juggling routine with both hands and feet, the timing, position and speed of the reactions in the activation of NO synthase must be precisely exact. Lu and He actually measured the dynamics of how calmodulin makes the process move along more efficiently. “It’s a significant advance and it provides direct experimental evidence and absolute numbers,” Stuehr said.

It was also gratifying to independently confirm that Stuehr’s lab’s measures and estimates had been very much on the right track. “This technique can be applied to all the other members of the family of NO synthase,” he said. The next paper will look at data from the blood vessel NO synthases. We can begin to understand why all the enzymes evolved to act so differently according to their biological functions. We’re very keen on trying it with others. We’ve figured out a lot of things in my lab over the last 20 years but there’s a lot of work to be done.”

Stuehr is energized by the potential of the connection with the BGSU lab. “It’s a neat combination. Peter’s and my expertise don’t really overlap,” he said of his collaboration with Lu. “We sort of speak different languages, so it’s been fun in that way, too. Now that he and Yufan have the technique and approach down, I feel that what we can accomplish is only limited by how much time they can spend on it.”

With Lu and Stuehr’s involvement, many other questions may soon be answered.