http://www.physorg.com/news95438056.html

Structural basis for photoswitching in fluorescent proteins brought into
focus

Graphic shows models of the on and off structural alignments of a
photoswitchable fluorescent protein. Credit: Courtesy S. James Remington

University of Oregon scientists have identified molecular features that
determine the light-emitting ability green fluorescent proteins, and by
strategically inserting a single oxygen atom they were able to keep the
lights turned off for up to 65 hours.
The findings, published online this week by the Proceedings of the National
Academy of Sciences, likely are applicable to most photoswitchable
fluorescent proteins, said S. James Remington, professor of physics and
member of the UO Institute of Molecular Biology.


"This new model makes specific predictions and improves the qualities of the
protein as a photo-switchable label," Remington said. "It gives us the first
picture of how these molecules can be switched on and off. That allows us to
design new variants to make the proteins more useful."

For more than a decade, fluorescent proteins - first isolated in jellyfish
and since found in a variety of colors from coral reef organisms -
revolutionized molecular biology, allowing scientists to use them as markers
for genetic expression, to locate molecules and observe activity within
cells.

The recent discovery of photoswitchable fluorescent proteins - which can be
manipulated with a laser - has been a significant development for cellular
research.

"Photoswitchable fluorescent proteins have tremendous advantages over
passive proteins," Remington said. "You can label all molecules but using a
laser under a microscope, you can activate only a small group of them. That
lets you follow the motion of subsets of molecules. We wanted to understand
the process, so that we can permanently switch them off and on or vary the
time delay."

However, he said, the mechanism of photoswitching was unknown, and in many
cases the proteins returned to their stable state randomly and
spontaneously.

Using a combination of rational mutagenesis and directed evolution, UO
doctoral student J. Nathan Henderson determined high-resolution crystal
structures of both the on and off states of a fluorescent protein isolated
from a sea anemone.

In the stable or fluorescent state of the molecule, two side chains of atoms
align in a coplanar fashion, flat and in orderly fashion. When hit with
bright laser light, the researchers observed that the protein rapidly went
dark as the rings rotated about 180 degrees and flip by some 45 degrees,
coming to rest in a non-coplanar and unstable alignment. The two structures
gave the researchers a chance to observe changes in the interactions between
neighboring groups.

Remington said that in the dark state, the molecule absorbs ultraviolet
light and doesn't emit any light at all. However, when the chromophore (a
group of atoms and electrons forming part of an molecule) absorbs
ultraviolet light, it occasionally ionizes and become negatively charged.
This causes the rings to flip back into the fluorescent state.

Having control of light emission would allow for more precise studies within
cells, he said.

Henderson studied the structures, noticing that in the dark state there was
an unfavorable interaction where carbon and oxygen atoms were adjacent to
each other. "Nathan looked at this and wondered what would happen if an
oxygen atom was inserted at a precise place," Remington said. "That would
make for a favorable interaction that stabilized the dark state. Based on
the structure, Henderson made a single mutation that delays the switch-on
time from five minutes to 65 hours.

Eventually, he added, the ability to control the on-off states could lead to
improvements in optical memory, such as single molecule information storage,
in addition to enhancing microscopic work and molecular labeling.

Source: University of Oregon
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