Scientists genetically encode IR probe

David Shenkenberg

Finding out how a protein interacts with other molecules in a cell can be challenging. Fluorophores such as rhodamine cannot label a specific protein. Although genetically encoded fluorophores such as GFP can identify a particular protein, they are often excited with UV radiation, a spectral region that frequently causes natural cellular components to also emit light. Therefore, microscopists have to determine whether they are viewing their protein of interest or something else. In contrast, cells do not usually radiate when they are excited with IR light.

A nitrile chemical group strongly absorbs IR radiation, is sensitive to its local environment and is small, so it is not likely to interfere with normal cellular processes. Researchers at The Scripps Research Institute in La Jolla, Calif., determined whether it could be expressed in a specific region of a protein in E. coli. They also performed in vitro experiments to determine if it could function as a probe.

Initial experiments demonstrated that E. coli mutants selectively incorporated the probe into a site within a protein. That success inspired the investigators to produce mutant myoglobin with a nitrile group in its active site in E. coli. After they extracted and purified the protein, they examined it with a Bruker Fourier transform infrared (FTIR) spectrometer. Overall, the spectra for the mutant and normal myoglobin were similar both in the presence and absence of iron, suggesting that the probe does not disturb the normal function of myoglobin. Further FTIR analysis of myoglobin with oxygen, nitric oxide, carbon monoxide and iron indicated that the probe can discriminate between each chemical.

The researchers report these findings in the Nov. 1 issue of the Journal of the American Chemical Society. They believe that the probe can be used to study protein folding, molecular interactions, conformational changes and the electric field around proteins. “The electric fields produced in folded proteins influence nearly every aspect of protein structure and function,” said Roshan Perera, an author of the study.

The investigators are attempting the site-specific incorporation of the probe into two enzymes, human aldose reductase and dihydrofolate reductase, and they plan eventually to study protein conformational dynamics using 2-D IR spectroscopy.