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Chromophore isomer stabilization is critical to efficient fluorescence in Cyan Fluorescent Proteins Guillaume Gotthard, David von Stetten, Damien Clavel, Marjolaine Noirclerc-Savoye, and Antoine Royant Biochemistry, Just Accepted Manuscript • Publication Date (Web): 17 Nov 2017 Downloaded from http://pubs.acs.org on November 18, 2017
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Biochemistry
Chromophore isomer stabilization is critical to efficient fluorescence in Cyan Fluorescent Proteins Guillaume Gotthard†, David von Stetten†, Damien Clavel‡, Marjolaine Noirclerc-Savoye‡ & Antoine Royant*,†,‡ †European Synchrotron Radiation Facility, F-38043 Grenoble, France ‡Univ. Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, F-38000 Grenoble, France *
[email protected]; phone: +33 476 88 17 46 ABSTRACT: ECFP, the first usable Cyan Fluorescent Protein, was obtained by adapting the tyrosine-based chromophore environment in Green Fluorescent Protein to that of a st tryptophan-based one. This 1 -generation CFP was superseded by the popular Cerulean, CyPet and SCFP3A that were engineered by rational and random mutagenesis. Yet, the latter CFPs still exhibit suboptimal properties of pH sensitivity and reversible photobleaching behavior. These flaws were rd serendipitously corrected in the 3 -generation CFP mTurquoise and its successors without obvious rationale. We show here that the evolution process had unexpectedly rend modeled the chromophore environment in 2 -generation CFPs so as to accommodate a different isomer, whose formation is favored by acidic pH or light irradiation, and which emits fluorescence much less efficiently. Our results illustrate how fluorescent protein engineering based solely on fluorescence efficiency optimization may affect other photophysical or physicochemical parameters, and bring novel insights on the rational evolution of fluorescent proteins with a tryptophan-based chromophore.
genesis with the goal of optimizing FRET to a Yellow FP. Most of its mutations are located on the outside of the protein, leaving I167A as the sole mutation responsible for the changes in photophysical properties. nd
While improved compared to ECFP, the 2 -generation CFPs are not ideal: (i) their fluorescence efficiency is only of ~50%, (ii) their pKa, which is the pH at which half the fluorescence emission signal is lost, are rather close from physiological pH, and (iii) a significant increase of reversible photobleaching, i.e. the transient loss of fluorescence capacity 8,9 under illumination. While we have already attempted to 10,11 we here understand the rationale for the first drawback, nd investigate the structural response of 2 -generation CFPs to both acidification of the medium and light irradiation. Table 1. Key mutations in successive generations of CFPs. Position
65
72
145
146
148
167
175
ECFP3
T
S
Y
I
H
I
S
Cerulean4
A
A
SCFP3A6
A
T
he genetically-encoded fluorescent chromophore of GFP made it an appealing target for numerous engineering efforts that started after the sequencing and subsequent 1 cloning of GFP from Aequorea victoria. Those efforts try to match the various requirements of cell biologists, who eventually need to have a given fluorescent protein (FP) optimized for certain photophysical parameters including molar extinction coefficient (EC), fluorescence quantum yield (QY), fluorescence decay behavior, maturation speed, photobleaching or photochromism resistance, but also physico-chemical 2+ ones such as pH or concentration in small solutes (Ca , Cl ). Cyan FPs (CFPs) were developed on the basis of the Y66W mutant of GFP, in which the central residue tyrosine of the 2 chromophore is exchanged with a tryptophan . Mutations around the chromophore allowed for the accommodation of the increased bulk of the tryptophan residue, which led to ECFP, with a significant, yet modest level of fluorescence 3 (QY=0.36). In the mid-2000s, several independent initiatives 4 led to improved CFPs. First, Cerulean (QY=0.49) (Tables 1 and S1) was developed based on the first crystallographic structure of ECFP that showed two distinct conformations of 5 6 the protein near the chromophore. SCFP3A (QY=0.56) was obtained through a more systematic approach of mutagenizing residues near the chromophore. Noteworthy, Cerulean and SCFP3A share the critical mutation H148D at a location separating the chromophore from the bulk solvent region. 7 Finally, CyPet (QY=0.41) was engineered by random muta-
A
mCerulean313
S
A
14
S
mTurquoise211
S
Aquamarine
0.49
D
G A
S
D A
G
G
1a
F
D
0.56
7
0.41
1a
0.84
5a
0.80
G A
QY 0.36
D
CyPet7 mTurquoise12
Others
0.89 G
1a
0.93
a
include the monomerizing A206K mutation shown not to be necessary in most CFPs.15,16
The prior determination of the structures of Cerulean at 17 mildly acidic pH (pH 5.0) at 2.0 Å resolution and at near10 physiological pH (pH 7.0) at 1.15 Å resolution revealed the details of chromophore-protein interactions (Figure 1A). We have improved the resolution of the pH 7.0 Cerulean structure to 1.02 Å, which allowed us to visualize the vast majority of protons on the chromophore and neighboring residues, providing the directionality of hydrogen bonds (Figure S1). At pH 7.0, the chromophore is in the Z,Z configuration as in ECFP (Figure S2A). The chromophore is stabilized by a strong hydrogen bond (2.8 Å) between the indole ring nitrogen (H-donor) of the chromophore and Ser205 (H-acceptor), which is engaged in a very strong H-bond (2.5 Å) with Glu222 (H-acceptor), a crucial residue in fluorescent proteins that is 18 necessary for proper maturation of the chromophore. Glu222 is also H-bonded to the first residue of the chromophore Thr65 (H-donor) and to a water molecule sitting next to the chromophore (2.6 and 2.7 Å, respectively). The fact that Glu222 is an acceptor of three H-bonds validates that it is deprotonated at physiological pH, as these bonds partici19 pate to decrease the effective negative charge. At pH 5.0 the chromophore is in the Z,E configuration with the indole ring
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Figure 2. (A) Absorption (lines) and fluorescence (dashes) spectrum of Cerulean (green) and Cerulean S205A (magenta) at pH 7.0 and Cerulean at pH 4.0 (blue) (B) X-ray structure of the chromophore and its environment in Cerulean S205A at physiological pH. The 2Fobs-Fcalc electron density was contoured at a 1.5 σ level around the chromophore (in magenta) and surrounding residues (in grey).
deprotonated aspartate (pKa of 3.9), but not in ECFP because a protonated histidine (pKa of 6.1) does not have any lone pair. CyPet also has a histidine at position 148, and the Z,E 20 isomer seen in its structure obtained at pH 7.0 can be rationalized by the fact that His148 is still singly protonated at this pH, and as such can provide a lone pair to stabilize the indole ring of the chromophore. Figure 1. (A) Chromophore isomers in Cerulean: (Z,Z) configuration at pH 7.0 (left, PDB code 2WSN10) and (Z,E) configuration at pH 5.0 (right, PDB code 2Q5717). (B) Chromophore configuration and environment in the acidic pH structures of SCFP3A (left, pH 4.5, Z,E configuration) and ECFP (right, pH 5.0, Z,Z configuration). The 2Fobs-Fcalc electron density is contoured at a 1.5 σ level around the chromophore (in green or cyan) and surrounding residues (in grey). (C) Absorption spectra of Cerulean, SCF3A and ECFP equilibrated at pH 8.0 (blue), pH 5.0 (red) and pH 3.0 (grey).
nitrogen H-bonded to Asp148 (Figure S2B). Ser205 appears to have a second conformation which is not H-bonded to Glu222 (Supplementary Methods). The latter residue is still H-bonded to Thr65 (2.6 Å), except that the side chain of Thr65 has rotated away by ~105°. We previously solved the physiological pH structure of 11 SCFP3A , and were curious to see if the presence of the H148D mutation would also trigger the same pH-induced chromophore isomerization. To this end, we solved the structure of SCFP3A at 1.56 Å resolution at the acidic pH of 4.5 (Figure S3). The structure shows a chromophore in the Z,E configuration with similar interactions with neighboring residues as in Cerulean, except that there is only one H-bond between Asp148 and the chromophore and that Ser205 is fully rotated away from Glu222 (Figure 1B, left). The last of nd the 2 -generation CFPs, CyPet, has recently been shown to undergo the same pH-induced isomerization, yet with a 20 much higher pKa, above 7.0. nd
Visualizing the pH-induced isomerization in all 2 generation CFPs raised the question of whether this phenomenon also occurs in their precursor ECFP. pH titration of absorption properties of Cerulean and SCFP3A from 8.0 down to 3.0 reveals a blue-shift before acidic denaturation (Figure 1C) 17 characteristic of the chromophore isomerization. However, ECFP does not show such a blue-shift, suggesting the absence of isomerization. This was confirmed indeed by our 1.29 Å structure of ECFP at pH 5.0, in which we observed the sole presence of the Z,Z isomer (Figure 1B, right). This can be explained by the fact that residue 148 can be a H-bond acceptor in Cerulean and SCFP3A thanks to the six lone pairs of a
While the Z,Z configuration of the chromophore is retained in the acidic pH structure of ECFP, its protein environment is th substantially affected at the location of the 7 strand, which adopts two distinct conformations (Figure S4A). The minor one (30% occupancy) closely resembles those observed at physiological pH (Figure S4B). The major one (70% occupancy) is rotated by ~180° vs. the minor conformation, bringing the side chain of Tyr145 from the bulk solvent to the interior th of the protein. In other words, the 7 strand can also adopt the conformation that it holds in the structure of GFP and the vast majority of its mutants (Figure S4C), reversing the structural effects of the initial mutations introduced to make 3,10 CFP fluorescent . This structure allows us to reconcile the discrepancy between the two previously published ECFP structures. Whereas the later one exhibits essentially one th conformation of the 7 strand with Tyr145 pointing outside 10 the protein (PDB code 2WSN), the earlier one has two conformations where Tyr145 is either pointing inwards for the major conformation (PDB code 1OXD) or outwards for the 5 minor conformation (PDB code 1OXE) (Figure S4D). A likely explanation is that the earlier structure was determined from crystals grown with PEG as precipitant without any buffering agent. Since PEG solutions tend to acidify over time, it is probable that this structure is of ECFP at slightly acidic pH, hence presents a mixture of two largely different conforth mations of the 7 strand. Realizing that the single H148D mutation had such a profound effect on chromophore stabilization over a large pH range, we decided to understand further the delicate balance of protein-chromophore interactions by mutating Ser205, the other residue capable of engaging in an H-bond with the indole ring. We characterized the S205A mutant of Cerulean (Cerulean-S205A) both by spectroscopic and crystallographic methods at near-physiological pH. Examination of the UVVis absorption and fluorescence spectra shows that the absorption and emission maxima are blue-shifted compared to those of Cerulean at pH 7.0 and are comparable to those of Cerulean at pH 5.0 (Figure 2A), suggesting that the chromophore is in the Z,E configuration in Cerulean-S205A. To confirm that the blue-shift is a marker of the chromophore con-
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Biochemistry phore in the excited state by rotation around the methylene 21 bridge).
Figure 3. (A) In crystallo fluorescence and (B) in crystallo light absorption spectra of Cerulean crystals under blue light illumination (440 nm laser). Arrows indicate the loss of fluorescence intensity and the blue shift in the absorption spectra. (C) X-ray crystal structure of the chromophore and its environment in Cerulean after 440 nm light irradiation for 15 min at a physiological pH. Colored dashed lines indicate the H-bond stabilizing network for each chromophore isomers.
figuration in Cerulean S205A, Cerulean and ECFP, we used non-resonant Raman spectroscopy (Figure S5). Indeed, the 1.16 Å resolution structure of Cerulean-S205A at pH 7.0 shows that the protein-chromophore interactions are very similar to those seen in Cerulean at acidic pH (Figure 2B). This result remarkably highlights the direct role of Ser205 in stabilizing the chromophore of CFPs in the Z,Z rather than in the Z,E configuration at physiological pH. Since CeruleanS205A contained 100% of chromophore in the Z,E configuration, it gave us the chance to study the spectroscopic properties of the Z,E chromophore, in particular to measure its EC and QY. We determined the EC and QY of Cerulean-S205A to be 44 -1 -1 mM .cm and 0.07, respectively, to be compared to the Ceru-1 -1 lean values of 30 mM .cm and 0.49. This means that the Z,E chromophore is able to absorb photons 40% more efficiently, but that these photons are converted 7 times less efficiently into fluorescent photons, resulting in an overall 5-fold loss of brightness. In a microscope, the loss is accentuated by the correlated blue shifts in excitation and emission maxima, which are sub-optimal when used with the classical CFP set of excitation and emission filters (usually centered at 436 or 440 +/-10 nm and 480 +/-20 nm, respectively). The fact that the chromophore is less fluorescent in the Z,E than in the Z,Z configuration can be rationalized by inspecting the interactions stabilizing the indole ring of the chromophore. In both isomers, the nitrogen is H-bonded to a single residue (either Ser205 or Asp148), and the bulk of the stabilization is provided by van der Waals (vdW) interactions with several aliphatic residues. In ECFP and Cerulean at physiological pH, vdW interactions from Val61, Thr62, Ile146, Phe165 and Ile167 have been shown to be critical in control10 ling the fluorescence efficiency of the Z,Z chromophore. The indole ring of Z,E configuration of the chromophore is less stabilized in acidic Cerulean or Cerulean-S205A as it loses Phe165 and Ile167 as vdW interaction partners, creating a cavity that could accommodate a distorted chromophore (Figure S6). In this case, the protein environment cannot efficiently hinder non-radiative de-excitation of the chromo-
Because Cerulean is the CFP that exhibits the maximum of 8 light-induced reversible photobleaching, we reasoned that illuminating a crystal of Cerulean with blue light would maximize the population of bleached molecules and make it detectable for structural analysis. First, in order to identify the spectroscopic characteristics of photobleached Cerulean, we exposed Cerulean crystals to pulsed blue light illumination at room temperature and monitored both its UV-vis absorption and fluorescence emission spectra over time. We observed a progressive decay of the fluorescence signal (Figure 3A) which was accompanied by a small, yet significant blue-shift and increase of the absorption peak (Figure 3B), suggesting the partial build-up of the Z,E species within the crystal. We then irradiated a crystal of Cerulean in its crystallization drop with a fiber connected to a 440 nm laser for 15 min. Fishing and flashcooling were performed under continuous illumination, and an X-ray diffraction data set was collected at 1.38 Å resolution. Structure modelling and refinement reveals a mixture of ~1/3 and 2/3 of isomerized and nonisomerized chromophores, respectively, and a model for photoconverted Cerulean was built around the Z,E chromophore taking into account the partial changes of surrounding residues. The structure of photoconverted Cerulean (Cerulean-PC440, Figure 3C and S7) shows a structure similar, but not identical to those of Cerulean at acidic pH and CeruleanS205A. The chromophore is in the Z,E configuration and Thr65 rotated away, but still in interaction with Glu222. The differences are on the conformation of Ser205, which is rotated away by almost 180° and on the H-bond acceptor of the indole ring nitrogen, which is a water molecule inserted inbetween the chromophore and the carbonyl group of Asp148, whose side chain remains in place. These results can be used to provide a structural basis for the mechanism of the reversible light-induced bleaching of Cerulean. Each time a photon is absorbed, the chromophore reaches the excited state S1, from which it usually decays via radiative (fluorescence) or non-radiative (internal conversion) pathways. We propose that in Cerulean, the chromophore can also isomerize to the Z,E configuration in the excited state S1, and relaxes to a new energy minimum of the ground state S0 (light-adapted chromophore) which has different fluorescent properties (increased EC, decreased QY) than the normal Z,Z configuration (dark-adapted chromophore). Because light-irradiation leads to a steady-state equilibrium between the dark- and light-adapted chromophores, only a fraction of the molecules present a light-adapted chromophore. This explains why the fluorescence decrease, the blue shift of the absorption maximum and the increase in absorbance are all only partial: most molecules are always in the dark-adapted state.
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Scheme 1. Configurational energy landscape of the Cerulean chromophore in the ground and excited states. The chromophore can isomerize in the excited state and relax to a level of slightly higher energy than that of the Z,Z configuration. The energy of the corresponding Z,E configuration well is decreased by acidification of the medium or single-point mutation near the chromophore.
Taken together, the four structures of Cerulean and SCFP3A at acidic pH, of Cerulean with the single-point mutation S205A or irradiated with blue light all show the same structural feature that is associated with a loss of cyan fluorescence: the isomerization of the chromophore to the Z,E configuration. The different nature of these perturbations indicates that the conformational landscape of Cerulean (and nd other 2 -generation CFPs) has gained the ability to accommodate this particular isomer of the tryptophan-based 22 chromophore , when compared to that of ECFP, and thus, to be less fluorescent in certain conditions. The key element in the evolution has been the introduction of Asp148 in Cerulean and SCFP3A, which provides an alternative H-bond acceptor than Ser205 to the indole ring nitrogen of the chromophore. In CyPet, which retains His148 from ECFP, the likely explanation resides in the I167A mutation which removes a strong vdW interaction with the six-membered ring of the indole ring present in ECFP, thus destabilizing the Z,Z configuration. The fact that photobleached Cerulean does not need the reorientation of Asp148 to stabilize the isomerized chromophore suggests that the same mechanism may occur in the reversible photobleaching of ECFP, yet at a 8,9 lower efficiency. In brief, the presence of a very strong Ser205-Glu222 Hnd bond controls the configuration of the chromophore in 2 generation CFPs. This H-bond can be perturbed by (i) the pH-induced reorganization of the H-bond network around the chromophore, (ii) the loss of the H-bond between residue 205 and the chromophore, and (iii) the decay from the excited state of the chromophore into another, energetically equivalent, well of the conformational landscape of Cerulean (Scheme 1). The first well corresponds to the Z,Z chromophore, a stable situation at physiological pH and low light level. The second one can be ascribed to a Z,E chromophore, populated by seemingly minor perturbations such as the protonation of a few key acidic residues, the loss of one hydroxyl group on a key residue or the relaxation from the excited state of the chromophore.
A third generation of CFPs has emerged in the 2010s, all exhibiting at least a 1.5-fold increase in fluorescence efficiennd cy compared to 2 -generation CFPs. These new CFPs were fortuitously cured from the aforementioned flaws as demon11 strated by the largely improved pKas (as low as 3.1) and the 8,11 lack of reversible photobleaching. The origin of the improvement is evidently the T65S mutation, which amounts to the removal of a single methyl group on the first residue composing the chromophore, since this single mutation is nd the only difference between the 2 -generation CFP SCFP3A rd and the 3 -generation CFP mTurquoise. However, the mechanism by which isomerization has been hindered is unclear and would deserve to be elucidated by a comprehensive study including molecular dynamics simulations. Finally, the insights developed in our study will find a broader application to the optimization of red-shifted fluorescent proteins based on a tryptophan-based chromophore such as the yellow and orange fluorescent proteins recently derived from 23 the monomeric red fluorescent protein FusionRed, but also 24 25 to the study of the cyan/green FPs WasCFP and NowGFP , whose anionic tryptophan-based chromophore exhibits a more pronounced photochromic behavior that needs to be explained by both chromophore isomerization and protonation mechanisms.
ASSOCIATED CONTENT Supporting Information Methods, supplementary tables and figures.
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected]; phone : +33 476 88 17 46.
ORCID Antoine Royant: 0000-0002-1919-8649 Damien Clavel: 0000-0002-3075-3228 Guillaume Gotthard: 0000-0003-2830-0286 Marjolaine Noirclerc-Savoye: 0000-0002-5481-6561
Notes The authors declare no competing financial interests.
Accession codes The structures described here have been deposited as PDB entries 5OX9 (SCFP3A at pH 4.5), 5OX8 (ECFP at pH 5.0), 5OXA (Cerulean-S205A at pH 7.0), 5OXB (Cerulean-PC440) and 5OXC (Cerulean at 1.02 Å)
ACKNOWLEDGMENT The authors acknowledge the ESRF for financial support, access to beamlines and facilities for molecular biology via its in-house research program. The authors also acknowledge the platforms of the Grenoble Instruct center (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) supported by the French Infrastructure for Integrated Structural Biology Initiative FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB).
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Chromophore isomer stabilization is critical to efficient fluorescence in Cyan Fluorescent Proteins Guillaume Gotthard, David von Stetten, Damien Clavel, Marjolaine Noirclerc-Savoye & Antoine Royant
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