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of the DNA sequence, separated by a short linker of four amino acids, to several
subcellular localization proteins [ 56 ]. Tandem dimer constructs have also been
developed with DsRed [ 54 ] and EosFP [ 57 ].
Other procedures for reducing FP oligomerization and aggregation include remov-
ing several basic residues from the N-terminus and simultaneous co-expression of
FP-tagged proteins with an excess of a nonfluorescent mutant of the marker protein to
generate heterodimers or heterotetramers that contain only a single target polypeptide
and can thus be considered pseudo-monomeric [ 58 ].
3.4 Photostability
Since practically all FP applications rely on fluorescence measurements, photo-
stability is a key parameter for the applicability of any FP. The photostability of a
chromophore is quantified by its quantum yield of photobleaching,
F b , which, for
an ensemble, is the ratio between the number of photobleached molecules and the
total number of photons absorbed within a certain time interval. Typically, the
fluorescent chromophore will emit 10 4 -10 5 photons until it falls victim to perma-
nent photodestruction.
Although insufficient photostability frequently limits the performance of a partic-
ular FP, this particular property has often only been an afterthought in FP optimiza-
tion. At present, the relation between structure and photostability is only poorly
understood. Therefore, to improve photostability, multiple rounds of (directed or
random) mutagenesis are typically performed, with screening for photostability
after each round. Tsien and coworkers noticed the importance of residue 163 in
influencing the photostability of mRFP1 variants [ 59 ]. For mTFP1, the Asn63Thr
mutation resulted in a particularly large increase in photostability [ 60 ]. For red FPs,
photobleaching probabilities of ~10 6 are currently being achieved [ 42 ].
3.5 Color Tuning
3.5.1 Modifying the Chromophore Environment
The p -HBI chromophore can exist as an anionic phenolate species or a neutral
hydroxyphenyl form, depending on pH and/or the local environment in its binding
pocket. Under physiological conditions, the neutral form with an absorption peak at
395 nm predominates in GFP; the absorption maximum of the anionic chromophore
is at 475 nm.
By amino acid modifications in the chromophore microenvironment, the equi-
librium between the neutral and anionic chromophores can be markedly changed.
Shifting the equilibrium toward the anionic form leads to bright, enhanced GFP
(EGFP) variants. Interestingly, in a particular GFP variant (photoactivatable GFP,
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