Studying the binding and unbinding of molecules could lead to better insights into the study of antibodies.
Typically, engineers spend their time trying to reduce vibrations in their work because the movements often inhibit a positive outcome or even worse, cause damage.
But Todd Sulchek, an associate professor in mechanical engineering at Georgia Tech, found just the opposite in fine-tuning an atomic force microscope for his research in detecting and measuring forces exerted by interacting molecules, a dance never before fully observed. Although not the primary focus of his work, adding electronic white noise to the instrument was critical to significantly increasing the precision of measurements deep within energy wells during molecular interactions.
The outcome of this engineering solution from Sulchek and graduate researchers Ahmad Haider and Daniel Potter was recently published in the Proceedings of the National Academy of Sciences.
Sulchek’s work focuses on the mechanical and adhesive properties of cell and biological systems and the development of microsystems to aid in their study. He has been interested in the binding and unbinding of molecules to provide insights potentially leading to widespread applications in many areas of the life sciences, everything from drug targeting to the study of antibodies to better understanding autoimmune disorders and immunotherapy to treat cancer.
Sulchek acknowledges “They are very hard to study,” even though the use of an atomic force microscope has gone a long way toward advancing understanding. Interaction between molecules takes place within a complex energy landscape, and while the kinetic behavior and nanoscale properties of the molecules have been measured, information about the underlying energy landscapes has been difficult to obtain. That’s because of the lack of a tool to adequately measure the interaction that takes place deep within. “You see how these molecules are bound together or unbound,” he says, but not the attachment or detachment itself that takes place at nanosecond speed.
Researchers can see that the attachment of molecules takes places at multiple sites rather than one very strong attachment site. They believe this allows more functionality of targeting. “That not only regulates how long they can stay attached but there are some dynamic aspects that can be an extra tool for modulating its effects,” Sulchek says. “You can, for example, turn it off by using an inhibitor to block the interaction whereas if you have a really strong bond, that wouldn’t be as feasible.”
They also believe that to fully come together, there is a two-step process: the interaction step and a twist step for locking. “A lot of protein is believed to interact in a similar way,” Sulcheck says.
A nanoscale, cone-shaped sensing probe used by an atomic force microscope is sensitive enough to detect forces exerted by interacting molecules and measures the attracting energies when placed near the molecules. The cone is attached to a tiny flexible stick that wiggles as the atomic forces push and pull the cone. This wiggling is transferred into the microscope and converted into a usable signal, illustrating what is called an energy well. The top of the well is where the adhesive forces are about to take hold and the bottom is about where the molecules meet.
While researchers can see the top and bottom of the energy well, the microscope cannot provide detailed measurements of increases and decreases of energy deep within the energy well, where the two-step process actually takes place.
Sulchek and his team met the challenge by adding electronic noise that allowed the probe to feel the interaction when it was still relatively far away from the surface of the molecules. The electronic vibration also weakened the adhesive forces bringing the stick and molecules together. These actions allow researchers to take more samples and longer ones. “Before it was either black or white, but now we’re succeeding at getting varying shades of gray,” Sulchek says.
Asked how he came up with the idea of adding more vibration, Sulchek says, “I wish it was completely my idea. Somebody else thought of it first a long time ago.” He recalls reading about it some time ago but didn’t appreciate what they were showing.
“What I’ve since realized is that by doing that you allow the cantilever, which is the force probe, to measure the deepest part of the energy well, which is what dictates whether something binds or doesn’t bind,” he notes. “When they add this little bit of noise, it seemed to increase. It wasn’t so important for their adhesion measurements but when I became interested in energy landscapes, it was the magic trick that makes it work.”
Now that the theoretical approach to how to add the noise, how much noise to add, and how to control different parameters of the measurement system for accurate measurements of the energy landscapes has been completed, the next step is to apply it to study biological interactions. One of the next goals is to be able to record the two separate steps of interaction and, longer term, identify the roles of the two binding sites that antibodies have.
“Right now, [this work] is a basic science tool. It’s a holy grail to be able to understand how these complicated molecules work,” he says. “We know they work by coming together and binding, but to understand it at a more fundamental level would be important for basic science.”
Nancy S. Giges is an independent writer.