New Technique Provides First Look At Atomic Interactions Between Proteins

BUFFALO, N.Y. -- In an experiment that they expect will prove applicable to the whole cascade of biological communications known as signal transduction, researchers at the University at Buffalo's Center for Structural Biology have for the first time produced and observed the separation of two kinds of atomic interactions between proteins.

They did it using a new device they developed, the first that combines a high-pressure capability with state-of-the-art, three-dimensional nuclear magnetic spectroscopy.

The cost of the new method is a fraction of that of other techniques that have been used in attempts to conduct similar experiments.

A paper describing the research was published in the Nov. 13 issue of the Journal of the American Chemical Society.

The research provides scientists with the first tool that will allow them to determine which kinds of interactions involved in the association of proteins are more necessary in a particular protein complex.

These protein-protein interactions are not only at the heart of many biological functions, but also are critical in the development of disease states, such as cancer.

"Proteins associate because of two kinds of interactions: ionic interactions involving the attraction of opposite charges and more subtle interactions often termed van der Waals" or "hydrophobic interactions," said A. Joshua Wand, Ph.D., professor of chemistry, biological sciences and biophysics at UB and director of the Center for Structural Biology. "Until now, it has been nearly impossible to cleanly determine which class of interaction or force is the dominant contributor to the association of two proteins."

The ability to determine which kind of interaction is more necessary is critical in drug development.

"If you're designing a drug, you need to know which is more important: the greasy, hydrophobic surface contacts that occur between two proteins or the ionic interactions between them," he said. "Now we may have a tool to routinely solve that question."

In the experiments conducted at UB, the researchers were able to selectively break the ionic interactions between two proteins while keeping the complex intact.

"What this means is that in this case, the ionic interactions that were broken were responsible for specificity, and were not necessary for general binding," said Wand.

Consequently, if pharmaceutical scientists were designing a drug that needed to bind one of these proteins, they would focus on compounds that would promote surface contact.

Wand said the research provides a way to settle a long-standing argument in chemistry over which kinds of interactions confer affinity, which confer specificity and whether or not they are different interactions or the same. Affinity determines the ability of two proteins to stick together, while specificity allows for recognition between them.

To find out, each interaction must be isolated and studied. But until now, scientists have not had a reliable method with which to do this.

Chemists and biologists have traditionally used mutagenesis, a process by which they genetically alter one amino acid in a protein complex to separate and study the two interactions.

The alteration of the amino acid has actually changed the proteins themselves, so that the results are complicated to analyze and often inaccurate.

Researchers also have tried to isolate the interactions in a protein complex by subjecting it to very high pressures. Ionic interactions are extremely sensitive to pressures of 500-600 atmospheres, or 10,000 pounds per square inch. Under these conditions, in contrast, hydrophobic interactions are much less sensitive to high pressure.

Therefore, Wand explained, pressure can be used to selectively break ionic interactions while leaving the hydrophobic interactions intact. "This allows one to see if ionic interactions are necessary for binding," he added.

The UB researchers were successful with the inexpensive device they developed, which combines state-of-the-art, three-dimensional nuclear magnetic spectroscopy, an atomic-scale technique, with high pressure.

"Advanced NMR techniques allow us to examine the structure of proteins in atomic detail under conditions where the ionic interactions can be removed by pressure," said Wand. "For the first time, we are able to get a detailed look at the structural role of ionic interactions between proteins."

Co-authors on the paper, all at UB, are Jeffrey L. Urbauer, Ph.D., research assistant professor of chemistry; Mark R. Ehrhardt, Ph.D., doctoral candidate in chemistry; Ramona J. Bieber, research specialist in chemistry, and Peter F. Flynn, Ph.D., research assistant professor of chemistry.