Surface plasmon resonance (SPR) is not the new kid on the block. It’s been an industry workhorse technology for more than 20 years. What is new is coupling this premier technology with thermodynamic methodologies or molecular dynamics simulations. When used together, these provide a powerful means to generate a greater depth of information than either can alone. Further, the ability to sensitively gauge kinetics and equilibrium information positions use of SPR as an adjunct to high-throughput screening and affinity ranking during lead optimization.
The recent “Developments in Protein Interaction Analysis” conference hosted by GE Healthcare highlighted several of these synergistic partnerships as well as new ways SPR is being employed in the modern-day drug discovery arena.
Typical SPR requires very little material and does not need an external probe for monitoring interactions. This label-free, real-time technology is initiated by immobilizing a component, such as protein or nucleic acid, onto a sensor chip. The other component(s) are then injected over the flow cell(s) in the desired buffer. SPR detection relies on a resonance interaction between the light beam and the thin metal film on the sensor chip.
Allostery is the scientifically intriguing process in which the binding of a ligand to a macromolecule alters ligand binding at distant sites. Named by Nobelist Jacques Monod in the early 1960s to describe the sigmoidal binding curve of oxygen to hemoglobin, its molecular mechanisms have been a holy grail of biochemistry ever since.
“Scientists have spent decades trying to understand protein allostery through ligand-binding kinetics, thermodynamics, and structures, but we still do not have a unified picture,” said Jannette Carey, Ph.D., professor of chemistry, Princeton University. Besides its academic interest, allostery is thought by some to offer a new approach to drug discovery.
Dr. Carey’s laboratory uses a combination of experimental and computational techniques to study allostery in several protein-ligand systems.
“We initially quantify a binding process using biochemical and biophysical approaches, including isothermal titration calorimetry (ITC) and SPR. ITC measures the heat flow associated with a molecular interaction, complementing SPR by providing thermodynamic information.
“Recently we added molecular dynamics simulations (MD) to our arsenal, in collaboration with Professor Rüdiger Ettrich of the Czech Academy of Sciences. MD applies Newton’s laws of motion to molecules, providing a temporal description of structural changes.
“I was stunned when we saw that MD was able to trace an allosteric response from specific atomic detail to global conformational change.”
Experimentalists, including Dr. Carey, initially were skeptical of MD. “MD can be used to make experimentally testable predictions, putting it firmly in the realm of the scientific method. Besides requiring huge computational power, MD interpretation is complex and subtle, and thus a job for experts. So don’t expect an app for that any time soon.”
Dr. Carey’s take-home message is that “quantitative studies of ligand binding are the first step in understanding allostery and the biological roles of molecular interactions. In every course I teach I try to work ligand-binding theory and practice into the curriculum.”