Figure 1.
Fluoroproline variants of EGFP.
(A) Characteristic β-barrel structure of EGFP with the 10 Pro residues highlighted. Cro66 indicates the fluorophore. (B) Chemical structures of proline and the two proline analogs, (2S,4S)-4-fluoroproline ((4S)-FPro), and (2S,4R)-4-fluoroproline ((4R)-FPro). (C) Expression profile of EGFP and its 4-FPro variants in E. coli. EGFP and (4S)-FPro-EGFP are predominantly soluble, whereas (4R)-FPro-EGFP is insoluble. Purified EGFP was applied as the molecular weight marker (M) and is indicated by the arrow; S, soluble protein fraction; I, insoluble protein fraction. Proteins were separated by SDS-PAGE and stained with Coomassie Brillant Blue.
Figure 2.
Stereo image of the crystal structure of (4S)-FPro-EGFP.
All Pro residues were replaced by (4S)-FPro. Fluoroprolines (13, 54, 56, 58, 75, 89, 187, 192, 196, 211) as well as the C- and N-termini (C, N) are indicated. The chromophore is shown in green and fluorines in cyan. Note that all fluorinated Pro residues except (4S)-FPro56 exhibit a Cγ-endo pucker. Only (4S)-FPro56 shows a Cγ-exo puckered pyrrolidine ring.
Figure 3.
Fluorescence recovery of EGFP and (4S)-FPro-EGFP.
The proteins were denatured by boiling (95°C, 5 min) in 8 M urea and refolded by 100-fold dilution into the buffer without urea (see Methods section for details). Fluorescence emission profiles of (A) (4S)-FPro-EGFP and (B) EGFP upon excitation of the chromophore at 488 nm before denaturation and after 24 h refolding at room temperature. (4S)-FPro-EGFP recovers more than 95% of its fluorescence before denaturation, whereas EGFP recovers only up to 60% of its initial fluorescence (this is in agreement with literature data). (C) The refolding kinetics of both proteins starts with an initial fast phase that is followed by a slow refolding phase. (4S)-FPro-EGFP refolds approximately 2 times faster than EGFP. The percentage of refolding was calculated on the basis of the final constant amount of fluorescence, corresponding to 100% of refolding. Normalized fluorescence in arbitrary units (au) was plotted against time.
Table 1.
X-ray data collection and refinement statistics.
Figure 4.
X-ray structure of the proline-rich pentapeptide (4S)-FPro54-Val55-(4S)-FPro56-Trp57-(4S)-FPro58 (PVPWP).
The continuous electron density (grey, 2Fo-Fc; contouring levels 1 σ) indicates fluorine atoms at the 4S-position in three buried Pro residues (54, 56, 58). Their experimental electron densities are localized unambiguously (image preparation with PYMOL (http://pymol.sourceforge.net/)). Out of the three Pro residues forming trans peptide bonds, only Pro56 exhibits predominant Cγ-exo pucker whereas the other two have pyrrolidine rings with Cγ-endo conformation. The rigid local secondary structure of this motif forces the (4S)-fluorinated pyrrolidine ring of (4S)-FPro56 into a stereochemically unfavorable Cγ-exo pucker.
Figure 5.
Local microenvironments of the fluorinated prolines in (4S)-FPro-EGFP.
The high resolution (2.1 Å) X-ray crystallographic structure of (4S)-FPro-EGFP allowed identification of new interactions introduced by 4S-fluorination. The fluorine atoms are characterized by well defined electron densities at the H→F replacement sites and facilitated unambiguous determination of the conformation of the pyrrolidine rings (see also Fig. 2). Fluorines are cyan, the new interactions are shown in yellow except one repulsive interaction, which is indicated in grey. All images were prepared with PYMOL (http://pymol.sourceforge.net/). (A) (4S)-FPro13 interacts with the backbone -NH- of Asp117 (3.46 Å) and with Oγ2 of Thr118 (3.03 Å) on the neighboring strand. (B) The fluorinated PVPWP motif: the 4S-fluorine of (4S)-FPro56 is in a stereochemically unfavored position; it is most probably involved in a repulsive interaction with the backbone carbonyl group of Asn153 on the neighboring strand (measured crystallographic distance: 3.07 Å). The other two fluorinated prolines are involved in dipole interactions with neighboring backbone -NH- groups: (4S)-FPro54 with Val55 (3.40 Å) and (4S)-FPro58 with Thr59 (3.17 Å). (C) (4S)-FPro75 interacts with the backbone -NH- of Met78 (3.32 Å) and establishes a contact (∼3.4 Å) with the -NH- of the His77 imidazole ring. (D) (4S)-FPro196 interacts with the backbone -NH- of the adjacent Ala154 (3.43 Å), and (E) (4S)-FPro89 with that of the succeeding Glu90 (3.46 Å). Finally, (F) (4S)-FPro211 interacts with both, the backbone -NH- (3.40 Å) and Nб (3.06 Å) of the succeeding Asn212. In total, the fluorine atoms in (4S)-FPro-EGFP establish 12 novel interactions that are absent in EGFP.