best FRET pair _2012 update

[offterdi1]

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Dear all,

This question has been asked in the past already. But the color palette of
XFPs has increased substantially in the meantime.
Which combination of FPs is considered the best FRET pair right now?
Maybe one should also seperate between acceptor bleaching, sensitized
emission (ie filter fret) and FLIM, since the demands are quite different
for the three methods.
Any input is very welcome!


Martin

[Tim Feinstein-2]

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Hi Martin,

From what I understand CFP/YFP and its close variants such as cerulean, venus, etc remain almost freakishly effective compared with most alternatives.  If there exists a green/orange or orange/red pair with similar efficiency and lack of unwanted artifacts (e.g., photoconverson) then it is news to me.   About why that is, maybe it has to do with the odd emission spectrum of CFP.  Or perhaps it involves some quantum whatsit that the folks at CERN can figure out now that they have found their Higgs boson.  

cheers,


TF

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Jul 20, 2012, at 6:15 AM, Martin Offterdinger wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> This question has been asked in the past already. But the color palette of
> XFPs has increased substantially in the meantime.
> Which combination of FPs is considered the best FRET pair right now?
> Maybe one should also seperate between acceptor bleaching, sensitized
> emission (ie filter fret) and FLIM, since the demands are quite different
> for the three methods.
> Any input is very welcome!
>
>
> Martin

[Periasamy, Ammasi (ap3t)]

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Hello
If you look at the literature, majority of the time Cerulean and Venus is used as a FRET pair. The CFP is not a good donor for lifetime since it provides double exponential decays.
Cerulean3 and mTFP also used as a donor and Venus as an acceptor. Venus and tdTomato is another good FRET pair too. Please see some of our publications for various FRET pairs
1. Sun, Y., Wallrabe, H. Booker, C., Day, R.N. and Periasamy, A. (2010) Three-color spectral FRET microscopy localizes three interacting proteins in living cells. Biophysical J. Vol. 99, 1274-1283.
2. Sun, Y. Booker, C.F., Kumari, S., Day, R.N., Davdison, M. and Periasamy, A. (2009)
Characterization of an Orange Acceptor Fluorescent Protein for Sensitized Spectral FRET Microscopy using a White Light Laser. J. Biomed. Opt. 14(5), 054009 (pp1-11).
3.Day, R.N., Booker, C. and Periasamy, A. (2008) The Characterization of an improved donor fluorescent protein for Förster resonance energy transfer microscopy. J. Biomed. Opt. 13: 031203 (pp1-9).
Hope this helps
ammasi

Dr. Ammasi Periasamy
Professor & Center Director
W.M. Keck Center for Cellular Imaging (KCCI)
Biology, University of Virginia
B005, Physical Life Sciences Building (PLSB)
90, Geldard Drive, Charlottesville, VA 22904
(Campus Mail - P.O. Box 400328)
Voice: 434-243-7602 (Office); 982-4869 (lab)
Fax:[phone]; Email:[hidden email]
http://www.kcci.virginia.edu/contact/peri.php
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Offterdinger
Sent: Friday, July 20, 2012 6:15 AM
To: [hidden email]
Subject: best FRET pair _2012 update

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Dear all,

This question has been asked in the past already. But the color palette of XFPs has increased substantially in the meantime.
Which combination of FPs is considered the best FRET pair right now?
Maybe one should also seperate between acceptor bleaching, sensitized emission (ie filter fret) and FLIM, since the demands are quite different for the three methods.
Any input is very welcome!


Martin

[George McNamara]

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Hi Martin,

CY11.5 (ECFPdelta11-LE-delta5Venus) is still the best FRET fusion
protein. Will be hard to get above its ~95% efficiency.Hopefully someone
will replace the ECFP with a current generation cyan (see previous
replies) with monoexpenonential fluorescence lifetime etc.  Most DsRed
tetramers have green subunits, but FRET efficiently to red subunits.

Optimizing FRET biosensors, depends in part on their dimerization
tendency (A206 vs A206K at Aequorea FP's interface). See:

ACS Chem Biol. 2010 Feb 19;5(2):215-22. Reversible dimerization of
Aequorea victoria fluorescent proteins increases the dynamic range of
FRET-based indicators. Kotera I, Iwasaki T, Imamura H, Noji H, Nagai T.
Research Institute for Electronic Science, Hokkaido University, Kita-20
Nishi-10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan.
Fluorescent protein (FP)-based Forster resonance energy transfer (FRET)
technology is useful for development of functional indicators to
visualize second messenger molecules and activation of signaling
components in living cells. However, the design and construction of the
functional indicators require careful optimization of their structure at
the atomic level. Therefore, routine procedures for constructing
FRET-based indicators currently include the adjustment of the linker
length between the FPs and the sensor domain and relative dipole
orientation of the FP chromophore. Here we report that, in addition to
these techniques, optimization of the dimerization interface of Aequorea
FPs is essential to achieve the highest possible dynamic range of signal
change by FRET-based indicators. We performed spectroscopic analyses of
various indicators (cameleon, TN-XL, and ATeam) and their variants. We
chose variants containing mutant FPs with different dimerization
properties, i.e., no, weak, or enhanced dimerization of the donor or
acceptor FP. Our findings revealed that the FPs that dimerized weakly
yielded high-performance FRET-based indicators with the greatest dynamic
range.
PMID: 20047338



On 7/20/2012 6:15 AM, Martin Offterdinger wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> This question has been asked in the past already. But the color palette of
> XFPs has increased substantially in the meantime.
> Which combination of FPs is considered the best FRET pair right now?
> Maybe one should also seperate between acceptor bleaching, sensitized
> emission (ie filter fret) and FLIM, since the demands are quite different
> for the three methods.
> Any input is very welcome!
>
>
> Martin
>
>    

[Joachim Goedhart-2]

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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/17925859

As 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/20081836
The mono-exponential decay has been reproduced independently:
http://www.ncbi.nlm.nih.gov/pubmed/21221430

mTurquoise 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/21559477

Recently we have published an improved variant (mTurquoise2):
http://www.ncbi.nlm.nih.gov/pubmed/22434194
It 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.html

Best 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: [phone]
Fax: [phone]
http://www.science.uva.nl/research/mc/Joachim