Interplay of two dyes in the biosynthetic fluorescent protein. Credit: A. Skerra/TUM |
Since the middle of the 1990s a bright green fluorescent
protein has been used in research laboratories worldwide. Protein designers at
Technische Universitaet Muenchen (TUM) in Weihenstephan have now taken the
existing fluorescent protein a step further: They have managed to incorporate a
synthetic amino acid into the natural protein to create a new kind of chimeric
fluorescent bio-molecule by means of synthetic biology. By exploiting a special
physical effect, the fluorescent protein glows in turquoise when excited with
ultraviolet light and displays up to now unmatched properties. The results are
in the Journal of the American Chemical
Society.
Proteins are the most important functional biomolecules in
nature with numerous applications in life science research, biotechnology, and
medicine. So how can they be modified in the most effective way to attain
certain desired properties? In the past, the modifications were usually carried
out either chemically or via genetic engineering. The team of Professor Arne
Skerra has now developed a more elegant combined solution: By extending the
otherwise universal genetic code, the scientists are able to coerce bacterial
cells to produce tailored proteins with synthetic functional groups. To put
their idea to the test, they set out to crack a particularly hard nut: The
scientists wanted to incorporate a non-natural amino acid at a specific site
into a widely used natural protein.
In bioresearch this protein is commonly known as GFP (green
fluorescent protein). It emits a bright green glow and stems originally from a
jellyfish that uses the protein to make itself visible in the darkness of the
deep sea. The team chose a pale lavender coumarin pigment, serving as side
chain of a non-natural amino acid, as the synthetic group. The scientists fed
this artificial amino acid to a laboratory culture of Escherichia coli bacteria. Since the team had transferred the
modified genetic blueprints for the GFP to the bacteria—including the necessary
biosynthesis machinery—it incorporated the coumarin amino acid at a very
specific site into the fluorescent protein.
This spot in the GFP was carefully chosen, explains
Professor Skerra: “We positioned the synthetic amino acid at a very close
distance from the fluorescence center of the natural protein.” The scientists employed
the principle of the so-called Förster resonance energy transfer, or FRET for
short. Under favorable conditions, this process of physical energy transfer,
named after the German physical chemist Theodor Förster, allows energy to be
conveyed from one stimulated pigment to another in a radiation-less manner.
It was precisely this FRET effect that the scientists
implemented very elegantly in the new fluorescent protein. They defined the
distance between the imported chemical pigment and the biological blue-green
pigment of the jellyfish protein in such a way that the interplay between the
two dyes resulted in a new fluorescent chimeric biomolecule. Because of the
extreme proximity of the two luminescent groups the pale lavender of the
synthetic amino acid can no longer be detected; instead, the typical blue-green
color of the fluorescent protein dominates. “What is special here, and
different from the natural GFP, is that, thanks to the synthetically incorporated
amino acid, the fluorescence can be excited with a commercially available
black-light lamp in place of an expensive dedicated LASER apparatus,” explains
Sebastian Kuhn, who conducted these groundbreaking experiments as part of his
doctoral thesis.
According to Skerra, the design principle of the novel
bio-molecule, which is characterized by a particularly large and hard to
achieve wavelength difference between excitation and emitted light, should open
numerous interesting applications: “We have now demonstrated that the
technology works. Our strategy will enable the preparation of customized
fluorescent proteins in various colors for manifold future purposes.”