A Multi-Million “Treasure Hunter” Is Serving the Duke Research Enterprise

If scientific discovery is the world's most precious jewel, did you know that Duke has a multimillion-dollar “treasure hunter”, that is extremely efficient and versatile at discovering these diamonds? Knowledge "hunters" and science "adventurers" from basic science departments, engineering, and, well, essentially any program at Duke can use it to answer the most intricate and important questions. In what part of our body does a drug work and why? What kind of impurities can be found in wetland waters? How is climate change altering our environment? What contaminants affect the strength of polymer structures? What pigments did the Renaissance painters use? How do proteins interact with one another and with RNA and DNA in order to carry out life’s most basic functions?

A Versatile Technology

From Biochemistry to Medicine, from the Pratt School of Engineering to the Nicholas School of the Environment, from studying cancer drug efficacy to investigating pigments found in the world's most famous artworks, and from Nobel Laureates to eager undergraduate learners – this versatile and transdisciplinary technology is ready to serve research and academic projects. The technology is called Nuclear Magnetic Resonance spectroscopy (NMR) and it is housed within the Duke NMR Center, located in the Levine Science Research Center and the French Science Center. "When the principle of NMR was first demonstrated in 1944, nobody could envision its full potential," said Dr. Ron Venters, who has led the Duke NMR Center since the founding Director and his mentor, Dr. Leonard Spicer, "a university professor by design" – as Dr. Venters proudly refers to him, retired three years ago. The Center, which was established in 1986, has become an educational and research essential for hundreds of trainees and scientists of various backgrounds from Duke and beyond. They are united by a common goal: The need to understand the three-dimensional structure and properties of molecules at the atomic level in order to answer research questions that could unlock the path to discovery.

The NMR Center provides seven "state-of-the-art, multi-million-dollar instruments" – as Dr. Venters describes them - where researchers can run their experiments for very affordable fees. One of the flagship instruments, developed in 1999, operates at 800 megahertz (18.80 tesla) and costs 3.7 million dollars. The newest is a 700 megahertz (16.45 tesla) 2.3-million-dollar instrument installed in 2017. The Center staff provides intensive training on instrument use, data analysis, and offers research advice. "We are not staffed in such a way that we can accommodate doing every NMR-based research project on campus, but we are proficient at training you to collect and analyze data yourself," said Dr. Ben Bobay, the Assistant Director of the NMR Center. "We are not a 'submit-your-sample' type of Center. We provide very performant equipment and show you how to use it for your project." Dr. Venters specified that it was Dr. Spicer's idea to design the Center as a resource that is readily available to as many groups as possible; he wanted to train researchers to use the equipment – a strategy that fulfills the Center's mission of building technical capacity and providing research mentoring to students and scientists across Duke departments and research teams.

Dr. Venter’s Research Focuses on Developing New Methods for NMR

One of Dr. Venters first NMR projects, in 1994, involved developing isotopic labeling strategies to produce fully deuterated proteins, which was an idea of his own imagining that became very useful for everyone in the field. "If you completely exchange protons for deuterons in a protein, the NMR properties of that protein change dramatically, allowing you to study larger, more complicated, systems," Ron explained.

Asked what his current research goals are, Dr. Venters modestly mentioned two projects; one in partnership with NIEHS to study the recognition determinants of RNA binding proteins and another with multiple investigators within Duke to develop new anti-fungal drugs. "Systemic fungal infections can be very dangerous for immuno-compromised people, such as those with organ transplants,” Dr. Venters said. Venters and Dr. Joseph Heitman, professor in Molecular Genetics and Microbiology, are studying the structure of the FKBP12 protein with the goal to suppress it in a way that kills systemic fungal pathogens without affecting the human host. They are trying to improve upon an existing drug, FK506, also known as Tacrolimus, which is known to efficiently kill fungal pathogens, but which also causes immune suppression in the host. “We only could accomplish the goals of research projects likes these with NMR spectroscopy," Dr. Venters explains.

For those who aren’t well-versed in spectroscopy, here is how NMR works. Once study samples are placed in a strong magnetic field, nuclei resonate and behave like small magnets. The frequency at which each individual nucleus resonates in the magnet is unique and conveys extremely detailed information, about the surrounding atomic environment in a protein, a polymer, or other form of matter. "Instead of looking through a microscope to examine a protein, because it would be too small to see, we use NMR to “see” the three-dimensional structure of a protein at the atomic level," Dr. Venters said.

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NMR, a Universal Tool With an Astounding Array of Applications Discovered by 7 Nobel Prize Winners

"It is impossible to describe what NMR can do in one sentence because it has so many diverse and invaluable applications. When the principle was discovered, no one would have guessed how universal NMR would become," Dr. Venters emphasized. NMR evolved from a physics curiosity into an invaluable technique for applications in chemistry and medicine – where it also gained popularity, with the widespread Magnetic Resonance Imaging (MRI), which is based on the principles of NMR, is used to see detailed images of the body. There have been five Nobel Prizes awarded for research directly related to NMR between 1944 - when physicist Isidor Isaac Rabi discovered the "resonance method for recording the magnetic properties of atomic nuclei" - and 2003 when medical researchers Paul Lauterbur and Sir Peter Mansfield discovered magnetic resonance imaging. An additional six Nobel Awards have been given for research that contributed indirectly to the development of NMR, all in Physics.

NMR Technology Is Precise, Detailed, and Non-Destructive

Among the technologies used to investigate chemical and macromolecular structures, NMR is the winner in terms of precision, detail, and versatility. "NMR technology is effective because it achieves atomic resolution and is non-destructive to the sample. This is very important for researchers working with limited samples because they can recover their precious material and use it for further research," said Dr. Bobay. "What this technique does, and no other technique can do, is to look at the motion of proteins and how ligands bind to those proteins in their native liquid environment. The other structural techniques are done in a crystal phase," Dr. Venters added.

"NMR is an indispensable tool for chemists because it allows us to piece together the puzzle of how individual atoms are put together in a molecule, and how those structures change during a reaction,", said Dr. Katherine Franz, Alexander F. Hehmeyer Professor and Chair of the Duke Chemistry Department. Dr. Franz explained that her team is now trying to create new antibacterial agents that would selectively kill bacteria that cause infection without depleting the good bacteria that are part of a healthy microbiome. "If you've ever needed to take more than one course of an antibiotic to fight off an infection, or seen terrible outcomes when antibiotics fail, then you are aware of the serious and growing problem of drug resistance and the harmful consequences of the overuse of antibiotics. We are using NMR to tackle this problem by loading bacterial cells directly into NMR tubes, adding our molecules, and watching in real-time how different strains of bacteria change the structure of those molecules. I have to admit, it's pretty cool to see these data! We can see what is happening chemically in these cells, then correlate those changes with which cell types are killed by the treatment. That's how we connect chemical structure to biological activity," Dr. Franz described.

Dr. Zhou’s Lab Uses NMR To Develop New Antibiotics

Dr. Pei Zhou, professor of Biochemistry and Chemistry, is researching antibody development in the context of antibiotic resistance. "Pharmaceutical companies are not incentivized to develop new antibiotics. We try to fill this gap," Dr. Zhou explained. His laboratory has been working with the NMR Center on several projects in the past years and has published a number of papers as a result. "We found that people use crystallography in particular when trying to obtain high-resolution structures. But that technology provides a single static picture. With NMR technology we were able to capture the dynamics of an inhibitor in multiple confirmations, which is incredibly powerful. We have used the information to design better inhibitor compounds and more powerful antibiotics. Some of these compounds are now under commercial development," said Dr. Zhou.

An Educational Facility

"Our Center is as much an educational facility as a service facility," saidDr. Venters, NMR Center Director, who wants to expand upon the traditional user pool. The traditional user profile is comprised primarily of researchers in Chemistry and Biochemistry who rely upon NMR for their daily research activities, Dr. Venters reported. "In Chemistry alone, there are over 60 graduate students a year trained by Ben," added Dr. Venters.  With over 20 years of experience in protein structure determination and protein-small ligand interaction, Dr. Bobay has also leading the training for the NMR Center’s users. But first, tt was Dr. Venters who taught him how to collect, process and analyze NMR data for protein structure calculations. Dr. Bobay’s current research focuses on developing computational methods aimed at designing better ligands to be used as drugs. "Within the last year, we computationally screened almost 2500 peptides designed to purify a particular protein from blood serum. The computational method informed my collaborators to produce only a small subset of these peptides to test for their ability to purify this protein and, of this subset, most ended up being quite specific to that protein and competing well with current commercial ligands," explained Dr. Bobay.

Dr. Bobay is leading the NMR Center’s training at both locations, but especially at the 'walk-up' instruments, located in the French Family Science Center, where students drop in daily with their samples. Dr. Venters is providing support mostly at the central LSRC facility, designed more for longer-term macromolecular experiments and users from Biochemistry and other basic science departments.

Dr. Franz Is Using NMR To Study an Antimicrobial Peptide Found in Saliva

Dr. Franz, the chair of the Chemistry Department, recalls how important it was to receive support and expertise from the NMR Center staff while executing different experiments related to one of her research projects which tries to understand the "chemical changes that happen to an antimicrobial peptide that is naturally found in saliva." She added: "This has been a challenging puzzle to solve because data collected from different kinds of experiments don't seem to fit together, at least not at first. The NMR Center staff helped us get better definition around each puzzle piece. It's starting to fit together now, and we are on our way to connecting chemical structure to biological activity."

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Experiential Learning for Graduate Students Resulted in a Scientific Paper

The NMR Center staff believe strongly in experiential learning. The Center recently helped several graduate students led by Dr. Matt Scaglione, Assistant Professor in Molecular Genetics and Microbiology. Dr. Scaglione's lab is interested in the regulation of protein homeostasis (proteostasis) in neurodegenerative diseases. "One of Matt’s graduate students was reading articles about what other researchers had done and realized that they needed to collect NMR data. We trained these students to do the experiments, helped them with sample preparation, with data analysis and they are in the process of publishing a paper as a result of the interaction with our Center," said Dr. Venters. "NMR was critical to our studies as other techniques would not have been able to detect the changes that we could observe by NMR," said Dr. Scaglione.

Dr. Scaglione’s research focuses on understanding molecules in the brain that can cause or prevent neurodegenerative diseases. "Currently the therapies for neurodegenerative diseases are limited. In our lab, we believe that if we can understand how molecules in the brain cause or prevent neurodegeneration, we will be able to devise new therapies to treat these diseases. While studying changes in a protein that causes neurodegeneration we realized that the changes likely resulted in the protein adopting a different shape. Over the past year, we have been working with the NMR Center to identify the changes in this protein's conformation. We also did experiments that demonstrated that we can correct the protein’s shape, a finding that has led to drug discovery efforts in the lab," added Scaglione.

"We would love to help more groups that might not even realize that their project is a perfect NMR project," Dr. Bobay added. He and Dr. Venters hope that more groups will reach out to the Center to request training and support for research projects. "Essentially, anybody who is doing basic research or who is currently taking classes in basic sciences at Duke could potentially benefit from utilizing NMR," said Dr. Venters.

"There is so much to gain from having time on the instruments in addition to the theoretical knowledge that is being taught in the classroom," Dr. Bobay explained. "Students from my class are taught the physics behind NMR and are then given homework to sit down on an NMR instrument and collect data that demonstrate these basic principles. We treat it like a cooking show," jokes Dr. Bobay: "Even if the students don’t collect perfect data, we grab the ideal data from underneath the table and give it to them: here's some good data for you to analyze." Dr. Matt Scaglione has only praise for the NMR Center's team: "Working with Ron and the rest of the NMR Center has been exceptional. Everyone in the NMR Center is very helpful in helping us design, run, and interpret our experiments. The ability to send graduate students to the Center, with no NMR experience, and have them collect data within the next day or two has been critical for streamlining our research." The NMR Center has also conducted outreach efforts with the North Carolina School of Science and Math, helping high school students get a glimpse into the world of sophisticated University-level research.

The NMR Center has also supported efforts within the research community in the Research Triangle Park. "We feel that we have an ethical obligation to reach out and support the local community and not live sequestered within our ivory tower," Dr. Venters said. He continued: "it is not only in the interest of startups that cannot afford to buy multi-million-dollar NMR instruments, but it is also important for us, to be exposed to research that is being conducted outside of Academia." Both Dr. Venters and Dr. Bobay truly believe in the societal impact of their work. They are in the process of starting a new research project focused on examining the structure of a protein responsible for the replication of a DNA virus causing massive destruction in plants and animals. "In the late 1990s, 90% of the crops in Uganda were devastated by this virus. Globalization brought the virus to the US which affects primarily porcine and agricultural industries," Dr. Bobay said. "There are known peptide inhibitors of this destructive virus. We will use NMR and computational methodologies to change these peptides in a rational, data-driven manner making them more specific to the viral protein and, subsequently, better inhibitors of it. Adding to the urgency, the current vaccine for this virus is becoming less effective, as a result of mutations", Dr. Bobay added.

Both Dr. Bobay and Dr. Venters will continue to support Duke's research efforts from the NMR Center. “I can think of no other technique as versatile or invaluable to science as a whole,” Dr.  Venters concluded.