Folding proteins in a cell
URBANA, Ill. – When it comes
to protein folding, cells have a few tricks up their sleeve, report researchers
from the University of Illinois at Urbana-Champaign. Using laser-based techniques,
they have shown for the first time that cells strongly modulate the speed and stability
of protein folding.
“This provides an entirely new mechanism by which cells
can control what their proteins do,” said Martin Gruebele, professor of chemistry,
physics and biophysics. “In addition to folding reactions, other reactions
are likely also to be modulated in the cell.”
Gruebele said reactions affected by the cellular environment might
be those related to Alzheimer’s, Huntington’s or mad cow diseases, all
of which are characterized by protein misfolding and plaques. Other reactions that
could potentially be affected because they take place inside a cell are those related
to the complex protection mechanisms that arise during a fever.
The cube shown here represents a magnified pixel imaged from the
cell, with folded (right-pointing arrow) and unfolded (left-pointing arrow) proteins
in equilibrium. The yellow structures are red acceptor and the green are donor fluorescent
labels, used to measure distances and the protein state. Courtesy of Martin Gruebele,
University of Illinois, Urbana.
The discovery was made possible because Gruebele and his colleagues
successfully combined laser-driven temperature-jump relaxation with fluorescence
microscopy. Although both techniques are individually well developed, merging them
presented challenges. One was how to spike the temperature up or down rapidly and
then keep it constant afterward on a large microscope stage with multiple objectives,
slides and other heat-conducting equipment.
The solution to this and other engineering problems, as outlined
in a paper published in the April 2010 issue of Nature Methods, involved multiple
lasers operating at different wavelengths. For temperature jumping, the researchers
used a laser emitting at 2.2 μm, employing two types of pulses. For one, they
shaped the laser pulse into a spike followed by a plateau, which caused a rapid
temperature rise. For the other, they preheated the cell and then dropped the laser
power to zero, achieving a downward temperature jump that took less than 50 ms.
They monitored the temperature profile by exciting an acceptor
dye with a green diode laser, calibrating the observed intensity to an absolute
temperature scale. They could do this on a pixel-by-pixel basis for the captured
When studying protein-folding dynamics, they used a blue LED or
an argon-ion laser to excite a donor attached – such as the acceptor –
to the biomolecule being studied. Thanks to Förster resonance energy transfer,
the resulting emission provided a molecular ruler to measure the distance between
donor and acceptor.
With optics, they captured the fluorescence, split it into red
and green channels and imaged it using a CCD camera, recording a frame every 16.7
ms. This was the limit for the time-resolution of the equipment. A faster camera
could cut that into the microsecond range, the researchers asserted.
In a series of experiments, the investigators looked at protein
folding of the phosphoglycerate kinase construct in two human cell lines at various
temperatures. They did this in vitro, or in the equivalent of a test tube, and in
a live cell.
The ability to do these studies in a live cell is new and led
to some interesting results, Gruebele said. “We showed that the protein is
more stable in the living cell than it is in the test tube, and that the protein
folds at a different rate in the living cell than in vitro.”
The reason for the differences has to do with the crowded nature
of the cell, he explained. In a test tube, a protein can fold without constraint.
A cell, in contrast, is full of structures that act as channels and barriers that
affect the rate of folding or keep it from happening altogether.
Using the technique, clinical researchers might be able to get
useful information about misfolding diseases, heat-shock responses or other medically
interesting cellular behaviors. As for the Urbana group, they are applying the technique
with a final outcome in mind, Gruebele said.
“Our ultimate goal is a better understanding of how temperature
affects the behavior of cells and organisms, at the cell and chemical level.”
- fluorescence microscopy
- Observation of samples using excitation produced fluorescence. A sample is placed within the excitation laser and the plane of observation is scanned. Emitted photons from the sample are filtered by a long pass dichroic optic and are detected and recorded for digital image reproduction.
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