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Phe99Ser). Mutations close to the chromophore may enhance the efficiency of
chromophore formation, whereas the more distant mutations Val163Ala and
Ser175Gly are likely to prevent protein misfolding at elevated temperatures; muta-
tion Met153Thr may improve solubility by decreasing the surface hydrophobicity
[ 40 ]. Additional protein engineering yielded the “superfolder GFP”, which displays
improved tolerance toward circular permutation, greater resistance to chemical
denaturants, and enhanced folding kinetics [ 41 ].
For other FPs, sometimes only a few amino acid replacements have been
observed to enhance protein expression at elevated temperatures. For example,
the Ile57Val and Phe102Leu mutations endowed eqFP611 with reliable expression
at 37 C, as was revealed by random mutagenesis with subsequent screening of the
resulting clones [ 42 ]. The monomeric version of EosFP (mEosFP) could even be
rendered thermotolerant by introducing a single point mutation, Ala69Val [ 43 ].
3.2 Cellular Toxicity
Aggregation of FPs is a general problem that may cause cytotoxic effects similar to
the toxicity associated with aggregate formation in neurodegenerative disease [ 44 ].
For example, Link et al. [ 45 ] have enhanced aggregation of GFP by adding a specific
16-peptide sequence to the C-terminus, which caused paralysis upon expression in
body wall muscle cells of C. elegans . Interestingly, optimized codon usage signifi-
cantly reduced the cytotoxicity of EosFP when expressed in murine stem cells,
indicating that also rather unspecific effects of overexpression might account for the
cytotoxicity of some FPs [ 46 ].
Under illumination, FPs produce toxic reactive oxygen species (ROS) [ 47 ]
including singlet oxygen, 1 O 2 . It attacks preferentially aromatic and sulfur-contain-
ing amino acids of proteins, but other cellular components might be affected as well.
For many applications, the phototoxicity of typical FPs is low enough to be ignored,
which is not surprising considering that FPs evolved in organisms exposed to O 2 and
plenty of sunlight.
KillerRed, however, is an engineered FP with an increased production of ROS.
It has been created by mutagenesis of a nonfluorescent chromoprotein [ 48 ]. Kill-
erRed is orders of magnitude more phototoxic than GFP. Ever under mild illumi-
nation conditions, this genetically encoded photosensitizer is capable of killing
most cells within minutes. KillerRed can also inactivate an enzyme to which it is
joined in a fusion construct. This property is exploited in a technique termed
“chromophore-assisted light inactivation” (CALI) [ 49 ].
3.3 Monomerization
Essentially all naturally occurring FPs discovered to date tend to form oligomers.
For a range of applications, the oligomeric nature of an FP is entirely irrelevant,
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