Posted by
Joachim Goedhart-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/best-FRET-pair-2012-update-tp7578698p7578707.html
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Hi Martin,
This is what I can offer from my experience.
Indeed, one should distinguish the different methods to measure FRET. In
addition, there is a difference between probes for measuring protein-protein
interactions and probes for biosensors. In case of biosensors, dimerization
tendency can be beneficial and is a variable that should be analyzed and
optimized (in some sensors it has a significant influence, in other sensors it
does not play a role). However, for measuring protein-protein interactions the
probes should be strictly monomeric (i.e. non-interacting).
For the three different methods:
(1) sensitized emission (ie filter fret)
-High QY acceptor. Especially the high quantum yield of the acceptor of
importance, as it determines the amount of sensitized emission. The high
absorbance and high QY of YFP make this probe and excellent acceptor for
FRET and hence (partly) explain the popularity of the CFP/YFP FRET pair. This
is in contrast to the rather poor QY of red FPs and explain their limited use as
acceptors in FRET pairs (another explanation is of course that monomeric Red
FPs were developed later in time). The best donor in combination with YFP is
mTurquoise2, see below.
(2) FLIM
-High QY, high lifetime donor and acceptor with high absorbance. Note that QY
of the acceptor is not important as only the lifetime of the donor is analyzed.
CFP/YFP is a good choice (with mTurquoise2 as a donor, see also below).
However, shifting FRET pairs to the red part of the spectrum generally
increase the FRET efficiency due to an increase of R0. We, and others, have
shown that yellow and orange FPs are excellent donors for FLIM-based FRET
measurements:
http://www.ncbi.nlm.nih.gov/pubmed/17925859(3) acceptor bleaching
-High QY photostable donor, acceptor needs be bleachable. So CFP/YFP can be
used with this method, but also yellow-red pairs can be succesfully analysed
by this method:
http://www.ncbi.nlm.nih.gov/pubmed/17925859As for CFP variants:
-We have published the first variant with a mono-exponential fluorescence
decay, mTurquoise, in 2010:
http://www.ncbi.nlm.nih.gov/pubmed/20081836The mono-exponential decay has been reproduced independently:
http://www.ncbi.nlm.nih.gov/pubmed/21221430mTurquoise shows excellent performance in FRET studies. For instance we
have shown that for a cAMP sensor, replacing the ECFP by mTurquoise
seriously improved performance in ratiometric- and FLIM-based FRET
measurements:
http://www.ncbi.nlm.nih.gov/pubmed/21559477Recently we have published an improved variant (mTurquoise2):
http://www.ncbi.nlm.nih.gov/pubmed/22434194It has the following characteristics:
-highest QY measured for a monomeric fluorescent protein (QY = 0.93)
-mono-exponential decay with a lifetime of 4.0 ns
-highest photostability in the CFP spectral class (emits on average 1.6 million
photons).
-low pKa of 3.1
-highest brightness of any CFP in eukaryotic cells
Based on the high QY (and hence high R0) mono-exponential lifetime and high
photostability, we claim that mTurquoise2 is the best cyan fluorescent protein
for FRET to YFP.
The plasmid encoding mTurquoise2 is available under MTA from our laboratory
(just write me an email), see also:
http://wwwmc.bio.uva.nl/Joachim/Resources-DNA.htmlBest Regards,
Joachim.
Dr. Joachim Goedhart
Section Molecular Cytology
Swammerdam Institute for Life Sciences
University of Amsterdam
Science Park 904
Room C2.264
NL-1098 XH Amsterdam
The Netherlands
Tel: +31(0)20 525 7774
Fax: +31(0)20 525 7934
http://www.science.uva.nl/research/mc/Joachim