Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/best-FRET-pair-2012-update-tp7578698p7578706.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy*****
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
>
>