- Scientists genetically encode IR probe
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
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.
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