IR difference spectroscopy reveals protein/pharmaceutical action in detail

Sept. 4, 2012

Bochum, Germany--Researchers at the Ruhr-Universität, Bochum (RUB) have developed a new IR spectroscopy technique to study the interaction between pharmaceuticals and their target proteins.

John Wallace

IR spectra (top left) provide information about structural changes in proteins. To study proteins that are activated through ligand binding, the RUB researchers anchored the molecules to a lipid bilayer (gray) via a His-Tag. The lipid bilayer itself is bound to a germanium crystal (IRE). IR light (red) is totally internally reflected in the crystal and attenuated by the anchored proteins. The researchers investigated the switch protein Ras (upper right), which plays a decisive role in cell growth.

Bochum, Germany--Researchers at the Ruhr-Universität, Bochum (RUB) have developed a new IR spectroscopy technique to study the interaction between pharmaceuticals and their target proteins.¹The method will be used to investigate pharmacological agent-protein interactions in a European Union project supported by various major European pharmaceutical companies.

In the technique, called IR difference spectroscopy, researchers follow dynamic processes in proteins. Previously, such processes could be observed in light-activated proteins, but not in proteins that are activated by binding with ligands—however, the latter is important, because it is how many illness-relevant molecules are activated. To analyze 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 IR spectroscopy with a surface-sensitive technique (attenuated total reflectance) and so-called “His-Tagging” (anchoring proteins to the measurement surface). In use, IR light is sent through a germanium crystal with proteins anchored on its surface; the light is totally reflected within the crystal, but partially absorbed by the proteins on the surface.

The His-Tag

This bonding of the proteins to the crystal succeeds through usage of the His-Tag, a simple amino acid chain commonly attached to proteins to enable their biochemical study. One big advantage is that an abundance of proteins are already fitted with the His-Tag, and therefore examining them using the new method is straightforward.

The RUB team first tried their new method on the switch protein Ras, which is the central on/off switch for cell growth. Defect, or oncogenic, Ras is one of the cells most frequently responsible for causing cancer. The researchers succeeded in fastening Ras to the measurement surface with the His-Tag, and then activating the Ras by binding it to a ligand. “The technique is so sensitive that we could resolve the signal of a five-nanometer-thick protein layer. That’s about 1/10000 of the diameter of a human hair,” says RUB researcher Jörn Güldenhaupt.

REFERENCE:

P. Pinkerneil et al., ChemPhysChem (2012), doi: 10.1002/cphc.201200358.

About the Author

John Wallace | Senior Technical Editor (1998-2022)

John Wallace was with Laser Focus World for nearly 25 years, retiring in late June 2022. He obtained a bachelor's degree in mechanical engineering and physics at Rutgers University and a master's in optical engineering at the University of Rochester. Before becoming an editor, John worked as an engineer at RCA, Exxon, Eastman Kodak, and GCA Corporation.