Grenoble, 20 March 2012: Scientists have designed a molecule which emits turquoise light more efficiently than seen before in living cells.
This improves the sensitivity of imaging of biological processes inside a living cell. The results have been published in Nature Communications on 20 March 2012.
IMAGE: An artistically inspired visualisation of the three-dimensional X-ray structure of the cyan fluorescence protein mTurquoise2 (Credit: Nature Communications/von Stetten/Royant/Goedhart).
The lead author of the publication is Antoine Royant from the Institut de Biologie Structurale/CNRS-CEA-Université Joseph Fourier in Grenoble. The team
also comprised scientists from the Universities of Amsterdam and Oxford, and
the European Synchrotron Radiation Facility (ESRF) in Grenoble.
Cyan fluorescent proteins (CFPs) are very popular in cell biology where they
are used to make processes visible inside a living cell, like in a movie. These
processes include the changes in the shape of large biological molecules. Since
the early 1990s, fluorescent proteins have become one of the most important
tools used in the biosciences and have helped the observation of previously
invisible processes such as the development of nerve cells in the brain or the
spread of cancer cells. The 2008 Nobel Prize in Chemistry crowned their
discovery and rapid development.
CFPs allow mapping of many processes in living cells when they can be attached
to a protein involved in an interaction or a conformational change. The CFP
inside the cell is localised by illuminating the cell with blue light which
makes the fluorescent protein emit light of a characteristic colour, which is
cyan for CFPs. The emission of light from the CFP reveals the target of
observation, the protein to which it is attached. However, these molecules have
long suffered from a weak fluorescence level, converting merely 36% of the
incoming blue into cyan light.
To achieve higher brightness, and with it improved sensitivity of fluorescent
imaging, the scientists based in France, led by Antoine Royant, teamed up with
colleagues from the Netherlands and the United Kingdom.
First, using highly brilliant X-ray beams at the ESRF, the teams from Grenoble
and Oxford uncovered subtle details of how CFPs store incoming energy and
retransmit it as fluorescent light: they produced tiny crystals of many
different improved CFPs and resolved their molecular structures. These
structures revealed a subtle process near the so-called chromophore, the
light-emitting complex inside the CFPs, whose fluorescence efficiency could be
modulated by the environment. “We could understand the function of individual
atoms within CFPs and pinpoint the part of the molecule that needed to be
modified to increase the fluorescence yield” says David von Stetten from the
ESRF.
In parallel to this work, the Amsterdam team led by Theodorus Gadella used an
innovative screening technique to study hundreds of modified CFP molecules,
measuring their fluorescence lifetimes under the microscope to identify which
had improved properties.
The result of this rational design is a new CFP, called mTurquoise2. By
combining structural and cellular biology efforts, the researchers managed to
show that mTurquoise2 has a fluorescence efficiency of 93%, unmatched for this
type of protein.
The new molecule will allow life scientists to study protein-protein
interactions in living cells with unprecedented sensitivity. High sensitivity
matters in processes where only a few proteins are involved and signals are
weak, and in fast reactions where the time available for accumulating
fluorescent light is short.
“With the new protein, many studies can now be performed with levels of
accuracy and detail that were impossible yesterday. Moreover, thanks to this
novel approach taking into account the structural dynamics of the protein,
scientists now hope to design improved fluorescent proteins emitting light of
different colours for use in other applications”, concludes Antoine Royant.
Reference:
J. Goedhart et al., Structure-guided evolution of cyan fluorescent proteins
towards a quantum yield of 93%, Nat. Commun. (2012); doi: 10.1038/ncomms1738.
http://www.esrf.eu/
