Synthetic Biology of Autofluorescent Proteins - Fluorescent Proteins: From Understanding to Design - page 113

Pro residues (see Fig. 6 ) of which nine form trans and only one (P89) forms a cis
peptide bond. Since GFPs are in general rigid proteins, it is reasonable to assume
that besides the cis / trans peptidyl-proline bond, Pro ring puckering plays a struc-
tural role [ 48 , 49 ].
However, this was not obvious from studies on peptide and small proteins since
they are usually very flexible in solution. For example, in these model systems
(4 R -F)Pro additionally stabilizes the trans conformation of peptidyl-proline bonds
(since 4( R )-fluoroproline favors C

- exo puckering). Similarly, (4 S -F)Pro stabilizes
the cis conformation due to the favored C

g

- endo puckering. In addition, Renner
et al . demonstrated that 4-fluorine containing Pro48 stabilizes/destabilizes barstar in
dependence of the enantiomer used [ 50 - 53 ].
However, the role of Pro-puckering in folding of complex protein structures
is for the first time identified in EGFP. In particular, EGFP[(4 R -F)Pro] is a
nonfluorescent insoluble protein, which could not be refolded. In contrast,
EGFP[(4 S -F)Pro] is a soluble fluorescent protein (Fig. 6 ). In solution, it is present
predominantly in the monomeric state, with some features superior to the parent
protein. For example, during the folding/refolding studies by fluorescence spec-
troscopy, it turned out that the recovery of fluorescence after denaturation/rena-
turation is much better for the variant (95%) compared to the parent protein
(60%). Also, the refolding kinetics is two times enhanced for the fluorine variant
(Table 3 ). In addition, the fluorinated variant of EGFP is less prone to aggregation
and crystallizes faster.
High-resolution three-dimensional structures of EGFP and EGFP[(4 S -F)Pro]
(PDB: 1EMG and 2Q6P) provided an explanation for these observations. A closer
inspection of the crystal structure of EGFP[(4 S -F)Pro] revealed that nine out of ten
(4 S -F)Pro residues show C

g

- exo
pucker. This side chain conformational preference of (4 S -F)Pro in the context of the
rigid GFP structure might be one of the main reasons for the enhanced refolding
properties. In other words, the proper stereochemical positioning of fluorine atoms
in the GFP protein matrix caused preorganization of the majority of its Pro-side
chains. Such subtle modifications, which confer extraordinary conformational
stability on a protein without perturbing its structure endow EGFP[(4 S -F)Pro]
with extraordinary properties [ 54 ]. This is further confirmed by the mapping of
12 new stabilizing interactions in the interior of EGFP[(4 S -F)Pro] up inspection of
its three-dimensional structure (Fig. 6 )[ 45 ].

g - endo puckering and only (4 S -F)Pro56 has a C g

Table 3 Comparison of the refolding kinetics and the fluorescence recovery of EGFP and
the superfolding EGFP[(4 S -F)Pro]. Upon incorporation of 4( S )-fluoroproline, EGFP exhibits
enhanced refolding kinetics as k for the fast phase as well as for the slow face is approximately
doubled. In addition, the fluorescence recovery is enhanced [ 45 ]
Protein

Refolding kinetics Fluorescence
Fast phase k 1 /(10 2 s 1 ) Slow phase k 2 /(10 2 s 1 ) Recovery after 24 h, RT

EGFP 1.41 0.15 60%

EGFP[(4 S -F)Pro] 3.01 0.36 > 95%