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was expressed only in insoluble (inclusion body) form. To overcome this problem,
they applied random mutagenesis and after 11 rounds - each screened several times
by FACS - they identified a mutant termed GFPm-11.3.3. This mutant tolerated Tfl
in its sequence and, in addition, after Tfl incorporation exhibited even higher
median fluorescence intensity than cells expressing GFPm with Leu. This protein
mutant capable of tolerating these substitutions has six original Leu positions
replaced by other canonical amino acids (L42I, L53I, L119I, L194R, L207I,
L221H). Four of them are distributed in the protein interior and two (194 and
221) are surface exposed. In addition, two new Leu residues (F46L, S202L) were
introduced into the protein's sequence; F46L is well known to increase chromo-
phore maturation rate in YFP [ 44 ].
The fluorinated GFPm-11.3.3 variant exhibits higher fluorescence intensities as
well as enhanced refolding rates [ 43 ]. This might be due to the fact that the CF 3
groups inside the protein enhance the hydrophobicity of the protein core. Thus,
composition refinement, which selected for tolerance toward the presence of fluor-
oalkyl groups in the hydrophobic core, leads to higher quantum yields (better
shielding of the chromophore), better expression yields, and solubility.
Another approach to improve the refolding properties of GFP is the incorpora-
tion of fluorinated proline derivatives (see Fig. 1 ), which was demonstrated in
EGFP [ 45 ]. In general, the refolding rate of GFP and its mutants is known to be
very slow (10-1,000 s) [ 37 ]. Such behavior is often associated with c is / trans
isomerization of peptidyl-proline bonds in protein structures [ 46 , 47 ]. Proline is
the only canonical amino acid with a secondary
-amino group due to its five-
membered pyrrolidine ring, which is directly incorporated into the protein's back-
bone. This ring can adopt two conformations, which differ in the position of C
termed C
- exo pucker and C
- endo pucker (Fig. 6 )[ 48 , 49 ]. EGFP consists of 10
Fig. 6 Effects of 4-fluoroproline enantiomers on the stability of EGFP. The primary sequence
of EGFP consists of ten Pro residues as indicated in the structure on the left . Substitution of
all these Pro residues with (4 R -F)Pro leads to the formation of unstructured inclusion bodies,
whereas the introduction of 10 (4 S -F)Pro residues yielded a protein with enhanced (re)folding
properties. Inspection of the crystal structure revealed 12 new stabilizing interactions after (4 S -F)Pro
incorporation. Two examples are shown on the right . Furthermore, with EGFP as model protein
the importance of Pro puckering for protein folding was identified for the first time in a complex
protein structure [ 45 ]
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