Surface-sensitive spectroscopy

Ruhr-Universität Bochum researchers have developed a new method for the detailed study of the interaction between pharmaceuticals and their target proteins.

“We now have a tool in our hands with which we can research the dynamics of pharmacologically interesting proteins in atomic detail,” Prof. Dr. Klaus Gerwert said.

“We want to undertake a targeted screening of substance libraries to look for potential pharmacological agents.” PD Dr. Carsten Kötting added that “with our technique future pharmaceuticals can be more closely tailored to illness-causing proteins, which can noticeably reduce the negative side effects of these drugs.”

With infrared difference spectroscopy, researchers follow dynamic processes in proteins.

For a long time, these processes could only be observed in light-activated proteins, but not in proteins that are activated by binding with ligands - but this is usually how many illness relevant molecules are activated.

To analyse the dynamics of such proteins, researchers have to fasten them to the measurement surface and pour a pharmacological-substance over them; the proteins can then interact with and be activated by this substance.

Even though this binding technique is possible, it cannot be used for all proteins. The RUB-Team worked around this problem by combining infrared (IR) spectroscopy with a surface-sensitive technique (attenuated total reflectance) and so-called “His-Tagging” (anchoring proteins to the measurement surface)

In conventional IR spectroscopy, an infrared beam is passed through a liquid sample; part of the light is absorbed by the proteins, which allows researchers to draw conclusions about their structure.

The RUB-researchers beamed the infrared light through a germanium crystal, on whose surface proteins were anchored. At the boundaries of the crystal the light is reflected over and over, thereby spreading throughout the crystal (attenuated total reflectance).

During this process, some of the light waves leave the crystal and reach the proteins that are fastened to its surface. A similar technique, the Surface Plasmon Resonance, is the standard for use in the pharmaceutical industry, but does not have the atomic resolution capabilities of the new technique.

This bonding of the proteins to the crystal succeeds through usage of the His-Tag, a simple amino acid chain, which is commonly attached to proteins today to enable their biochemical study - it is essentially a universal adapter.

Through the His-Tag the RUB-researchers were able to anchor the protein to the germanium crystal. As a result the molecules were firmly bound to the measurement surface, which transmits the infrared light to the proteins by the process of attenuated total reflectance.

The big advantage: an abundance of proteins are already fitted with the His-Tag; therefore examining them with the new method is unproblematic. All other proteins to which a His-Tag is attached can now also be accessed by IR spectroscopy. “This will help answer a multitude of biological and medical questions,” Gerwert said.