Biology Reference
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reporting the history of promoter activity and gene expression. Key amino
acids in the chromophore environment critical for the timing properties of
fluorescent timers have been determined via site-directed mutagenesis.
22
3.2. Photoactivatable fluorescent proteins
Another recent development is the discovery of fluorescent proteins with
light-modulated spectral properties, which are collectively termed “photo-
activatable fluorescent proteins,” and of fluorescent proteins that emit in the
far-red region and possess large Stokes shifts. These molecules have a number
of newapplications, includingmulticolor super-resolution imaging in fixedand
live cells.
41,42
In their resting states, photoactivatable fluorescent proteins (e.g.,
photoactivatable mCherries) are nonfluorescent, but they will emit bright red
fluorescence in response to UV or violet light illumination.
41
The structure
of the photoactivatable mCherry1 chromophore in the “dark state” is similar
to that of the mTagBFP chromophore, but this chromophore is not
fluorescent. Upon photon adsorption, the excited mTagBFP-like
chromophore is converted into a red chromophore via a Kolbe-like radical
reaction,
41,43
in a similar manner as the blue-to-red chromophore transition
(see
Section 2.2
).
3.3. Sensor for ions and small molecules
The original
A. victoria
GFP has several weaknesses, including dual peaks in
the excitation spectrum (395 and 475 nm) and pH-sensitive emission inten-
sity; these properties were improved during the development of enhanced
GFP. Using these properties, several fluorescent protein-based methods for
pH measurement have been developed, however.
44-46
In order to monitor
the acidic pH inside secretory vesicles, Dr. Rothman and colleagues
developed two classes of pHluorins
44
by substitution of amino acids
associated with the proton-relay network of Tyr66,
3,47-49
but not with
the chromophore itself, in GFP. “Ratiometric” pHluorin contains a
Ser202His mutation, along with eight additional substitutions, and
displays a reversible excitation ratio change between pH 5.5 and 7.5
(
R
395/475
¼
0.35-0.95), whereas “ecliptic” pHluorin gradually loses
fluorescence as the pH is lowered, at least until the pH reaches
6.0.
44
pHRed,
45
the first ratiometric, single-protein red fluorescent biosensor,
was recently engineered by introducing an Ala213Ser mutation into
mKeima, a red fluorescent protein with a long Stokes shift (excitation
and emission wavelengths of 440 and 610 nm, respectively).
50
pHRed
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