Role of Gln222 in Photoswitching of Aequorea Fluorescent Proteins: A

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Role of Gln222 in photoswitching of Aequorea Fluorescent Proteins: a twisting and H-bonding affair? Barbara Storti, Eleonora Margheritis, Gerardo Abbandonato, Giorgio Domenichini, Jes Dreier, Ilaria Testa, Gianpiero Garau, Riccardo Nifosì, and Ranieri Bizzarri ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00267 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 7, 2018

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ACS Chemical Biology

Figure 1. Absorption and fluorescence properties. (a) Absorption of wQ at pH 8.2 (blue) and pH 5.0 (red); fluorescence emission of wQ by excitation at 450 nm (black). (b) Lifetime decays of wQ (green) and wQT (black) at pH 8.2. (c) Emission spectra of wQ associated to the two lifetime components: τ1 (blue), τ2 (red) at pH 8.2; the dotted black line corresponds to steady state emission at the same excitation wavelength, 470 nm. 108x206mm (300 x 300 DPI)

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Figure 2. Reversible photoswitching properties of wQT. (a) Off-photoswitching: difference absorption spectra at different times upon illumination at 488 nm (0.3 W/cm2). (b) On-photoswitching: difference absorption spectra at different times upon illumination at 405 nm (3 mW/cm2) of the photoconverted form. (c) Comparison between the molar absorption of B state (blue line), the photosteady state (dashed green line), and the At state (red line). (d) Thermal recovery: difference absorption spectra at different times under dark at pH 7. (e) Thermal recovery kinetics at pH 6.7 (red line) and 7.1 (black line). (f) Thermal recovery: kinetic rate constant k vs. pH fitted to a single-site ionization titration curve (eq.1). 208x229mm (300 x 300 DPI)


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Figure 3. Photoswitching properties at high bleaching (488 nm) and reactivation (405 nm) illumination intensities. (a) Photoswitching cycles of wQ by alternating bleaching and reactivation illuminations; the colored scheme on top represents the illumination sequence (see Materials and Methods for details). (b) Average (500 cycles) off-switching kinetic curves of wQ at different bleaching powers: 0.88 kW/cm2 (red), 3.5 kW/cm2 (green), 10.6 kW/cm2 (cyan), 42.4 kW/cm2 (blue), and 41.3 kW/cm2 reactivation power (pulse of 50 µs); inset: comparison between wQ (blue) and wQT (black) at constant 42.4 kW/cm2 bleaching power, using log-scale for time to highlight differences between the off-switching rates. (c) Decrease in initial fluorescence along the switching cycles (fatigue) of wQ (blue), wQT (black), and rsEGFP2 (red) at constant 42.4 kW/cm2 bleaching power. 209x146mm (300 x 300 DPI)



Figure 4. Structure of wQ. (a) Ribbon representation of wQ (gray) with the internal chromophore (carbon atoms colored in green). (b) Local environment of the chromophore, consisting of a cyclized tripeptide made of S65, Y66 and G67, highlighting the presence of water molecules (red spheres) and hydrogen bonding interactions (black dashed lines). Labeled amino acids are in the one-letter code. The electron density overlaid on the chromophore and neighboring solvent molecules is also shown at a 1.5σ contour level (2fofc map, colored in blue). The alternate conformation of Ser65 (carbon atoms colored in yellow) partially interrupts the interactions involving V61, S65 and Q222. The sphere colored in cyan indicates the position of the water molecule present in the superimposed coordinates of the EGFP structure (PDB code: 2y0g), which mediates the hydrogen bond network in the vicinity of the chromophore. As shown by the electron density map, this water molecule is absent in the crystal structure of wQ. 197x105mm (300 x 300 DPI)


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Figure 5. a) Percentage of 500ps MDEX simulations leading to φ or τ twisting event (solid black), τ twisting event (shaded black and red), τ twisting event accompanied by 2Hb-Q222 conformation (shaded red). b) Instantaneous values of φ and τ during all MDEX simulations of wQ. The values for all snapshots are shown in gray and those with 2Hb-Q222 in red. The curves in the bottom graph show the percentage of snapshots containing 2Hb-Q222 as a function of τ for wQ, wQT and wQQ→E (see text). c) Ground-state (left) and excited-state (right) representative MD snapshots of wQ. 207x157mm (300 x 300 DPI)



Figure 6. qOLID images of GPI-wQ and COX8-EGFP expressed in CHO cells. a) Average stack intensity; b) map of photoswitchable fraction (fSW), corresponding to GPI-wQ; c) map of non-photoswitchable fraction (fNS), corresponding to COX8-EGFP; d) merge of fSW (red) and fNS green, highlighting the two intracellular localizations. Scale bar (panel a): 10 µm 128x37mm (300 x 300 DPI)


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Table of Contents 200x136mm (300 x 300 DPI)



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Role of Gln222 in photoswitching of Aequorea Fluorescent Proteins: a twisting and H-bonding affair? Barbara Storti,§ Eleonora Margheritis,¶ Gerardo Abbandonato,§ Giorgio Domenichini,§ Jes Dreier,‡ Ilaria Testa,‡ Gianpiero Garau,¶,* Riccardo Nifosì,§,* Ranieri Bizzarri§,* §

NEST, Scuola Normale Superiore and NANO-CNR, Pisa, Italy; ¶Center for Nanotechnology

Innovation @ NEST, Istituto Italiano di Tecnologia, Pisa, Italy; ‡Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Tomtebodavägen 23A 171 65, Stockholm, Sweden

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ABSTRACT

Reversibly photoswitchable fluorescent proteins (RSFPs) admirably combine the genetic encoding of fluorescence with the ability to repeatedly toggle between a bright and dark state, adding a new temporal dimension to the fluorescence signal. Accordingly, in the last years RSFPs have paved the way to novel applications in cell imaging that rely on their reversible photoswitching, including many super-resolution techniques such as F-PALM, RESOLFT, and SOFI that provide nanoscale pictures of the living matter. Yet many RSFPs have been engineered by a rational approach only to a limited extent, in absence of clear structure-property relationships that in most cases make anecdotic the emergence of the photoswitching. We recently reported [Bizzarri et al. J. Am Chem Soc. 2010, 102, 85] how the E222Q replacement is a single photoswitching mutation, since it restores the intrinsic cis-trans photoisomerization properties of the chromophore in otherwise non-switchable Aequorea proteins of different color and mutation pattern (Q-RSFPs). We here investigate the subtle role of Q222 on the excited state photophysics of the two simplest Q-RSFPs by a combined experimental and theoretical approach, using their non-switchable ancestor EGFP as benchmark. Our findings link indissolubly photoswitching and Q222 presence, by a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via H-bonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Thus Q222 belongs to a restricted family of single mutations that change dramatically the functional phenotype of a protein. The capability to distinguish quantitatively T65S/E222Q EGFP (”WildQ”, wQ) from the spectrally identical EGFP by



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quantitative Optical Lock-In Detection (qOLID) witnesses the relevance of this mutation for cell imaging.


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INTRODUCTION Fluorescent proteins (FPs) have revolutionized the in vivo imaging of cells and tissues, since they enable genetic encoding of bright visible fluorescence.1 Several variants with different spectral emission and photophysical properties were obtained by mutational strategies of the amino acid sequences of archetypal FPs.2 Development of FPs has recently led to the discovery of FPs that can be reversibly or irreversibly photoconverted between two optical states (phototransformable fluorescent proteins, PTFPs).3, 4 PTFPs add a temporal dimension to protein imaging techniques at the intracellular level with innovative application perspectives.5-7 Specialized PTFPs, the so-called Reversibly Photoswitchable Fluorescent Proteins, can be repeatedly photo-cycled between a fluorescent (on) and non-fluorescent (off) state on account of reversible conformational or chemical changes of the FP chromophore.8, 9 RSFPs are particularly interesting for all those advanced imaging strategies that make use of the dynamic nature of the fluorescence emission,10,

11

including super-resolution techniques such as PALM, STORM,

RESOLFT, SOFI, NL-SIM, which push the spatial resolution of fluorescence imaging beyond the diffraction limit.12-16 Even at conventional resolutions, RSFPs can strongly improve signal contrast, remove autofluorescence or yield a clean FRET image just by exploiting the alternate modulation of the fluorescence.17-19 With years, the number of RSFPs has increased from 1-2 to about 30-40 variants.8,

12, 20

In

most cases, valuable RSFPs were identified mostly by random mutagenesis of ancestors from different marine organism. Features relevant for the ultimate applications, i.e. the color, the brightness, the off-/on-switching rates or quantum yields, and the photochemical resistance to multiple cycles of fluorescence activation/deactivation guided this selection process. Surprisingly, only a few RSFPs developed so far belong to the Aequorea Victoria jellyfish



family,13,

14, 21, 22

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which still comprises the most popular FPs for biological and biophysical

studies.2 Direct optimization of RSFPs from Aequorea would enable easy incorporation of switchable markers on several hundred components of the library of eukaryotic proteins already in use.23 Recently, we described that a single point mutation in the primary sequence of Aequorea FPs, namely the E222Q substitution, transforms a non-switchable protein into a RSFP.22 The found photoswitching

mechanism

is

based

on

the

cis-trans

isomerization

of

the

4-(p-

hydroxybenzylidene)-5-imidazolinone (p-HBI1) chromophore coupled to a subsequent proton exchange of the chromophore with the surrounding residues (Scheme 1).22, 24 An intrinsic easy cis-trans photoisomerization of synthetic p-HBI25, 26 and similar analogs27 was proposed to occur for this and other RSFPs of non-aequorean origin.9, 28 Yet, our results indicate that the E222Q mutation is able to release this intrinsic chromophore property in otherwise non-switchable Aequorea variants. The role of E222Q substitution is intriguing from both a structural and a practical viewpoint. Structurally, E222Q belongs to that restricted number of single mutations that completely change the phenotype of a protein in the biological realm. Practically, E222Q affords a toolbox of RSFPs that can be easily applied to all Aequorea fusion constructs. Despite its relevance, the general mechanism of E222Q-induced photoswitching is still rather obscure, although its understanding may enable further rational mutagenesis of Aequorea variants, in order to improve RSFPs properties. In this work, we try to answer why a single amino acid replacement has such a profound effect on the protein photophysics. At the ground state, E222Q has been shown to stabilize at neutral pH the anionic form of pHBI.29, 30 Accordingly, we adopted as reference non-switchable protein the simplest Aequorea

1

We shall use p-HBI to denote the protein chromophore therefrom. 5 ACS Paragon Plus Environment

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FP that displays a similar protonation system around the chromophore, i.e. EGFP (F64L/S65T wtGFP). As RSFPs, we focus on the closest E222Q variants of EGFP preserving its spectral phenotype, i.e. wQ (“wildQ”: E222Q wtGFP or, equivalently, T65S/E222Q EGFP) and wQT (E222Q EGFP), without perturbing its monomeric state. wQ, wQT, and EGFP were subjected to spectroscopic and crystallographic investigations, and analyzed by molecular dynamics and computational approaches. Our results demonstrate that the photoswitchability brought about by E222Q is poorly related to structural changes at the ground state and to modifications of the emissive properties at the excited state. Instead, E222Q selectively stabilizes a subset of chromophore conformations at the excited state that are likely to promote cis to trans isomerization through a conical intersection. This highlights the subtle relevance of the E222Q single mutation, as well as its strikingly general effect.



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RESULTS The results are organized as follows: at first we describe the main absorption/fluorescence features of the “bright” state of the two switching proteins wQ and wQT, and we compare them with their non-switching benchmark EGFP. This helps to distinguish the spectroscopic features common to all mutants from those that depend on the specific mutations in position 65 and 222. Next, we present the spectroscopic characterization of photoswitching at low illumination intensity, thereby demonstrating how the absorption spectra can be used to recover the photoswitching yields by flash photolysis experiments. Then, we investigate the photoswitching behavior under the strong illumination intensities typical of a high-resolution microscope apparatus, in order to reveal the photochemical/physical resistance to several switching cycles under intense illumination power. The following sections dwell on the characterization of the photoswitching mechanism by molecular dynamics starting from the X-ray structure of WQ. Finally, we demonstrate the wide applicability of our photochromic proteins in the cellular imaging context by quantitative Optical Lock-In Detection (qOLID). Absorption and fluorescence properties in the native “bright” state. At pH ≥ 7, the absorption spectra of wQ and wQT in the visible region of the spectrum display a single large band centered around 480 nm (Figure 1a). This band corresponds to the B state, i.e. the chromophore deprotonated on Y66 hydroxyl function.29 The protonated form of the chromophore (A’ state) can be obtained by significantly decreasing the pH (Figure 1a, red line). Actually, pH titrations highlight that wQ and wQT have an ionization pKa around 6 (Table 1). EGFP is similarly characterized by a prevalent B band at pH>7 and pKa ~ 6 (Table 1). Nonetheless, the B bands of both wQ and wQT are somewhat blue-shifted (6-12 nm) compared to EGFP, although the extinction coefficients of the three proteins are very similar (Table 1).


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For all proteins, illumination on the high-energy tail (450 nm) of B leads to a green fluorescence emission that mirrors the absorption spectrum (Figure 1a). Similarly to absorption, wQ and wQT emission wavelengths are blue-shifted (3-4 nm) as compared to EGFP (Table 1). For other properties, however, wQ departs from a behavior common to EGFP and wQT. First, the emission quantum yields (Φ) of wQT and EGFP are nearly the same and about 20% higher than for wQ (Table 2). Second, the excitation spectrum of wQ contains a noticeable shoulder on the longer wavelength side, whereas this effect is much less pronounced in wQT and EGFP (Figure S1.1, SI1). Finally, wQT and EGFP show almost monoexponential fluorescence lifetime decays upon excitation at 470 nm, in contrast to the clear biexponential decay of wQ (Figure 1b, Table 1). Accordingly, we measured the emission spectra associated with the two lifetime components (Decay Associated Spectra, DAS) of wQ in the range 480-610 nm (Figure 1c). Both spectra peak around 505 nm, but the shorter component has a more pronounced emission in the blue 480-500 nm region than the longer one. These data suggest that wQ, differently from wQT and EGFP, is characterized by noticeable ground-state and excited-state heterogeneity, owing to a slow (with respect to lifetime) relaxation dynamics of residues around the chromophore. Photoswitching behavior in cuvette at low illumination intensity. Protein photoswitching was followed by steady-state absorption and fluorescence. At first, we addressed off-photoswitching (bleaching) of the B state by illumination at 488 nm (0.2-1 W/cm2). As expected, EGFP showed almost undetectable change in its absorption spectra upon illumination (Figure S1.2, SI1). Viceversa, the B state of Q222 variants was easily turned off into a state absorbing around 400 nm (Figure 2a). This dark state, hereafter denoted as At, can be identified with protonated p-HBI with trans configuration, according to the classical model of RSFP photo-isomerization developed by us and others (Scheme 1).22, 31 The same model predicts that the trans deprotonated 8 ACS Paragon Plus Environment


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chromophore (Bt) is not appreciably populated near physiological pH, owing to the significantly lower acidity of the phenolic proton in the trans configuration of p-HBI in protein.22 By illuminating at 405 nm, i.e. near At wavelength, the B state was promptly restored (Figure 2b). Notably, a much dimmer illumination intensity (2-3 mW/cm2) was required for efficient onphotoswitching (reactivation) of At as compared to turning off B. This suggests that the reactivation quantum yield (ϕon) is significantly larger than its off counterpart (ϕoff). This hypothesis was confirmed by the calculation of ϕoff and ϕon values by flash-photolysis experiments (Supporting information). For both Q222 proteins, ϕon was nearly 25-fold larger than ϕoff (Table 2). Additionally, the photoswitching yields of wQ and wQT are in good agreement with those found for other classical green RSFPs, including Dronpa, IrisFP, and the Q222 mutant Mut2Q developed by us.22 The reversible photoswitching behavior explains why prolonged illumination at 488 nm never leads to the complete disappearance of B. In fact, the kinetic analysis of the photo-isomerization system highlights that under continuous irradiation at any wavelength λ a photo-steady state is inevitably established where on- and off-photoswitching rates are equal.25 The photo-steady state At/B composition was calculated by analytical removal of the residual B contribution from the stationary absorption spectrum (Materials and methods). Residual B accounts for about 20-25% at photo-steady state when λ is 488 nm (Table 2). The same analysis affords the molar spectrum of At (Figure 2c). Interestingly, for both proteins the At maximum is red-shifted by 7-13 nm as compared to A’, although the extinction coefficients of the two states are very similar (Table 1). On-photoswitching is not the only process converting back At to B. Under dark conditions, At returns mono-exponentially into B with characteristic times (τc) ranging from hours to a few seconds (Figure 2d-e). This spontaneous trans→cis recovery is shared by many RSFPs and p9 ACS Paragon Plus Environment

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HBI alone.25 The recovery rate k (k = 1/τc) grows sigmoidally with pH (Figure 2f), according to a single-site ionization law:24 ( pKr − pH )    k0 + k∞ ⋅10 [1] k= ( pK r −pH ) 1+10

where k0, k∞, and pKr are the rate constants at low and high pH, and the pH at half-titration, respectively. Assuming that the single site ionization corresponds to the equilibrium At ↔Bt + H+, i.e. deprotonation of trans p-HBI favors thermal back-isomerization, pKr identifies with trans pKa.22 wQ and wQT share similar kinetic parameters (Table 2) and τc of 500-1,000 s at physiological pH. Photoswitching behavior at high illumination intensity The photoswitching behavior of wQ and wQT was evaluated at the illumination intensities (1-42 kW/cm2) typical of fluorescence microscopy. The proteins were embedded in a poly (acrylamide) gel at pH around 7 and an alternate sequence of off-switching (488 nm) and on-switching (405 nm) illumination pulses was applied while monitoring the fluorescence generated by a single focal spot (Figure 3a). Notably, the on-switching light intensity was optimized to provide maximum signal in the shortest available time lag, 20 ms. Figure 3b shows the average (on 500 cycles) off-switching kinetics for different blue light illumination intensities. In keeping with cuvette experiments, the offswitching rate increases at higher blue-light illumination intensity and the fluorescence reached a plateau once a photo-steady state is established. The contrast ratio D between initial and final fluorescence is larger for wQ than wQT (Table 2), as foreseeable by the higher amount of residual B at photo-steady state for the latter protein. Additionally, the off-switching rate is faster for wQ than wQT (Figure 3b, inset), in agreement with the larger ϕoff of wQ (Table 2). Remarkably, wQ and wQT showed radically different resistance (fatigue) to photoswitching



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(Figure 3c). At blue illumination intensities up to 18 kW/cm2, wQ loses only 5-10 % of the initial fluorescence after 500 cycles; this figure increases to 34% at 42 kW/cm2. Actually, wQ is almost indistinguishable from rsEGFP2 in terms of photoswitching fatigue (Figure 3c). At odds, the fluorescence of wQT dropped to less than 20% after 500 cycles for bleaching intensities larger than 3.5 kW/cm2, similarly to more delicate RSFPs such as Dronpa.15, 32 Crystal structure of wQ To investigate the photoswitching behavior of wQ and rationalize the observed spectroscopic properties, we determined the crystal structure of this protein to a resolution of 1.8 Å (Table S1.1). wQ displayed the traditional β-barrel structure with the chromophore located within the core of the protein (Figure 4a). The overall structure was essentially analogue to that of EGFP (PDB code: 2y0g),33 with a root-mean-square deviation in interatomic distances of 0.39 Å. In the vicinity of the chromophore the superimposition of the two structures showed that the hydroxyl group of residue S65 in wQ traced mainly the conformation of the hydroxyl group of the T65 in EGFP. This side-chain conformation assured in both structures the H-bond interaction with V61 carboxyl group, and simultaneously the interaction with the side chain of the residue in position 222 (Figure 4b). The electron density map showed anyway an alternative conformation with limited occupancy (~10%) for the S65– OH, which revealed an intrinsic mobility for this chromophore group in wQ. This alternate conformation partially interrupted the hydrogen bond network involving V61--S65--Q222 (Figure 4b). With respect to EGFP, the presence of Q222 in wQ had another main consequence on the chromophore environment: the absence of an internal water molecule (sphere colored in cyan in Figure 4b), which induced a relevant change of the solvent H-bond network around the chromophore. Contrary to E222 of the reported EGFP crystal structure, the side chain of Q222 in wQ assumed a unique conformation (Figure 4b).


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MD simulations Enhanced-sampling MD simulations of protein ground states were performed on the structures of the three mutants (wQ, wQT and EGFP). wQ and EGFP simulations stay generally close to their starting X-ray structure, though some side chains near the chromophore show conformation sub-populations. These features are described in detail in the Supporting Information (Section S2.1 and Tables S2.3 and S2.4). We also performed MD simulations using a model for the excited-state potential energy surface (MDEX), in order to monitor the excited-state dynamics and the passage through twisted conformations that enable radiationless decay and cis-trans photoisomerization (see methods and Supporting Information). From the (ground-state) MD simulations of wQ, wQT and EGFP we extracted 1200 starting structures and run 500ps of MDEX, monitoring the behavior of the methine bridge dihedral angles φ and τ (defined in Figure 5) and the change in hydrogen-bond network around the chromophore. Following Jonasson et al.34 we identify the conformations with φ,τ=±90 as those at which the fluorescence is quenched. In addition, values of τ around ±90 identify configurations amenable for cis-trans photoisomerization (in the cis planar conformation τ and φ are both 0, in the trans τ is ~ 180, whereas a 180 rotation around φ leads to the same cis chromophore). We first looked at how many simulations lead to at least one instance of φ or τ twisting, i.e. φ or τ getting larger than 90 in absolute value. The percentage is reported in Figure 5 for aggregate φ or τ twisting events and for τ twisting only in the three mutants (the wQQ → E case is discussed below). In wQ the chromophore reaches twisted configurations with a markedly higher efficiency (27% of the simulations against less than 6% in wQ and EGFP). These values can be related to the excited-state non-radiative decay time τNR (see Supporting Information, Eq. 1), yielding an estimated τNR of 1.6 ns, 7.9 ns and 12.0 ns respectively for wQ, wQT and EGFP. The 12 ACS Paragon Plus Environment


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MD simulations capture the excited-state trend in terms of fluorescence non-radiative lifetime (Table 1, entry τNR), though at this stage we cannot exclude that these stark differences are due to the T65 mutation common to EGFP and wQT. In the hypothesis that the prevalence of configurations at τ~±90 is correlated with the photoisomerization yield35 (and hence reversible photoswitching),2 our simulations correctly predict a much more efficient yield of wQ with respect to EGFP. They however do not discriminate between wQT and EGFP, which have a similar percentage of τ-twisted simulations. Nonetheless an intriguing role for Q222 emerges from further analysis of the H-bond network around the chromophore. The NH2 of the amide can form two simultaneous H-bonds. In the ground-state MD simulations it forms on average 1.2 and 1.4 H-bonds in wQ and wQT respectively mostly with the side chain of S205 and that of S/T65. We monitored the H-bond network around Q222-NH2 in the MDEX simulations, finding that, in several τ-twisted states, Q222-NH2 makes two hydrogen bonds, one to S205 and the other to cN2, the nitrogen of the chromophore imidazolidinone ring (hereafter we refer to this doubly bonded Q222-NH2 conformation as 2Hb-Q222). One representative snapshot containing this feature is shown in Figure 5c. The presence of the double H-bond is highly correlated with the τ-twisting events, as shown by the red bars in Figure 5a. These report the percentage of simulations showing at least

2

We should note that the configurations with τ~90 (70-110) mostly have φ in the -30-0 interval (Figure 5b). This

chromophore configuration closely resembles the intermediate state revealed in a very recent time-resolved femtosecond X-ray study of the off-on switching in rsEGFP2.36 Though the neutral chromophore state is the one involved in the off-on process, it is worth noting that their twisted chromophore structure is characterized by φ~0 and τ~90 (notice that our definition of φ is complementary to that in ref. 36).


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one τ-twisting event and, within ±0.5ps of the instant when τ reaches 90, the 2Hb-Q222 conformation. In order to establish this correlation, one needs to show that 2Hb-Q222 conformations are less frequent in planar or φ-twisted chromophore states. Panel b of Figure 5 reports in gray the instantaneous values of φ and τ during all MDEX trajectories of wQ. We first note that τ-twisting exclusively takes place by increasing the angle (in the same direction of the arrow in Figure 5c), while for φ-twisting the opposite direction is mostly adopted. In the same panel the red dots refer to MDEX snapshots containing 2Hb-Q222. These are 3.6% of the total in wQ and 0.6% in wQT (Table S2.5). The fraction of snapshots featuring 2Hb-Q222 to the total as a function of τ (bottom of Figure 5b) clearly reveals that 2Hb-Q222 is starkly over-represented at τ-twisted states. In the planar chromophore configuration cN2 is almost never contacted by Q222 because of steric hindrance by S/T65 and by the chromophore phenolate (Table S2.3). When τ increases towards 90 the imidazolidinone ring swinging brings cN2 closer to Q222 NH2, allowing an additional hydrogen bond to be formed. The emerging hypothesis is thus that the presence of Q222 selectively stabilizes twisted chromophore configurations eventually leading to cis-trans photoisomerization. E222 in EGFP can also be a H-bond donor to cN2, and can donate or accept a H-bond from S205 hydroxyl group. It cannot however act as a simultaneous H-bond donor to both groups. We monitored if the S05-OH → E222-OH→cN2 H-bond network has a role similar to 2Hb-Q222 and found that such network is never present in the MDEX simulations reaching the τ-twisted state.



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In the Supporting Information (Section S2.4) we analyze possible correlations between the conformations in the starting structures and chromophore twisting. Here we observe that the absence of T203-cO H-bond in the starting structure favors τ twisting (the opposite is instead true for φ twisting). In addition τ twisting in EGFP almost always happens starting from an anti E222 conformation. Clearly if the (ground-state) MD overestimates the population of these alternative conformations, then the efficiency of τ twisting is also overestimated. To better understand the role of Q222 in wQ we performed alchemical substitution, by setting to zero the partial charge of one of the Q222-NH2 hydrogen atoms, and assigning to the rest of the atoms the same charges as in E222. We kept the charged hydrogen as the one corresponding to the E222 anti conformation of the carboxyl group, which is the one almost always present in the EGFP MDEX simulations reaching τ twisting. We again performed 500ps-long MDEX simulations, starting from the ground-state wQ structures. In this way we avoid differences due to the ground-state starting structures and isolate the role of Q/E222 substitution over T/S65. As shown by the bars reported in Figure 5a (wQQ→E), the percentage of simulations reaching τtwisted states is remarkably reduced (from 14.3 to 5.1%), and very few 2Hb-Q222 conformations are detected (0.1%, Figure 5b and Table S2.5). This analysis shows that the double bond by Q222-NH2 has an active role in stabilizing τ-twisted states. The two-dimentional (τ,φ) excited-state force field used here is biased against the so-called hula-twist isomerization (i.e. a concerted torsion around τ,φ in opposite directions), which was initially ruled out for the chromophore in the gas phase based on ab-initio quantum chemistry approaches.36 Subsequent QM/MM studies of the chromophore in the protein environment have instead revived the role of hula-twist in the chromophore excited-state dynamics. For example Zhang et al.37 locates the conical intersection at τ=65, φ=-36 in the S65T/H148D GFP mutant, 15 ACS Paragon Plus Environment

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and Morozov et al.38 identifies the conical intersection leading to photoisomerization at around 90,-90. Based on these considerations we report in the Supporting Information additional simulations using a different excited-state force field, namely one forcing the chromophore to a hula-twisted conical intersection through simple harmonic restraints on the methine bridge dihedrals. These simulations let us draw the same conclusions stated above in terms of higher torsional mobility of the chromophore in wQ and in particular regarding the overrepresentation of the Hb-Q222 conformation in the proximity of τ-twisted states. Imaging applications of wQ. The interesting photoswitching properties of wQ prompted us to test this protein for Quantitative Optical Lock-In Detection (qOLID), a technique recently developed by us that takes advantage of RSFPs as biological markers.27 In short, different to classical OLID technique,17, 18, 39, 40 qOLID quantitatively separates the modulated and the nonmodulated signals deriving from photochromic and non-photochromic fluorophores emitting in the same spectral range, allowing for quantitative interpretation of overlapping signals.19 For this reason, qOLID has been applied to autofluorescence removal, two-probe signal separation, and photochromic FRET.19 In CHO cells, we concomitantly expressed wQ fused with glycophosphatidylinositol (GPI-wQ), and EGFP fused to the subunit VIII of human cytochromec oxidase (COX8) (Figure 6a). Then, we applied qOLID to quantitatively separate out the modulable GPI-wQ signal from the non-modulable signal of COX8-EGFP. The map of photoswitchable component fraction (fSW) clearly isolates the contribution of GPI-wQ, which is prevalently located on the plasma membrane as biologically expected (Figure 6b).41 Conversely, the map of non-photoswitchable component (fNS) fraction reproduces the specific staining of mitochondria, as COX8 targets specifically EGFP to these organelles (Figure 6c).42, 43 The twocolor merging map clearly distinguishes the localization of the two protein constructs (Figure 16 ACS Paragon Plus Environment


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6d). Notably, the fast and reproducible switching of wQ allows for very high precision of the fSW and fNS maps (0.4%, Figure S1.4, SI1).


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DISCUSSION Fluorescence is a highly dynamic phenomenon occurring in the ns-time scale. This relatively long time window allows for the occurrence of several photo-processes that can strongly modify the fluorophore properties, and hence the nature of emission. Outside the protein β-barrel, the photoexcited p-HBI undergoes a large structural twisting that quenches fluorescence through an effective internal conversion and, with significant yield (10-20%), enables the reversible cistrans isomerization of the molecule. Excited-state twisting is largely hampered in the tridimensional β−barrel structure of fluorescent proteins, restoring fluorescence emission and generally forbidding the photoisomerization. Nonetheless, in RSFPs the arrangement of residues around p-HBI subtly influences the excited state and allows the cis→trans photoisomerization to take place, albeit with a rather low quantum yield (10-4÷10-2). The reverse trans→cis photoisomerization, instead, occurs with higher yields (10-2÷10-1). In most RSFPs, photoisomerization of p-HBI is always followed by a proton exchange, enabling the reversible on-off cycling of the fluorescence between the anionic cis (bright) and neutral form (dark) of the chromophore. No clear correlation between the photoswitching behaviour and the protein sequence has been identified yet, except in one, intriguing, case: the E222Q replacement. Remarkably, E222Q was demonstrated by us to transform non-switchable Aequorea proteins of different sequences and colors into efficient RSFPs. We denote this family of photoswitchable proteins, Q-RSFPs. To understand the special role of E222Q mutation, we set out a comparative study between the two simplest Q-RSFPs, wQ (T65S E222Q EGFP) and wQT (E222Q EGFP) with their non-switchable common ancestor EGFP (S65T wtGFP). The study goal was to identify those structural and photophysical properties at the excited state that are uniquely associated with the E222Q replacement. 18 ACS Paragon Plus Environment


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The crystal structures of wQ and EGFP almost overlap, showing as main differences the absence of a water molecule proximal to p-HBI in wQ and different conformations for the side chains of residues in position 65 and 222. MD of the ground states, however, highlights that these structural features are likely transient at room temperature. Instead, previous X-ray data indicate that the carboxyl hydrogen of E222 flips between a syn and anti configuration in EGFP (Figure S2.2). Such configuration is fully caught by our MD simulations, which also indicate that p-HBI entangles the main surrounding residues via a rather constant number of H-bond in all mutants. Overall, these experimental and computational findings may explain why wQ, wQT and EGFP have closely related thermodynamic and photophysical properties at the ground state. The anionic cis p-HBI (B state) is largely prevalent in the three mutants at neutral pH and above, owing to pKa below 6. The absorption spectra are poorly dependent upon the primary protein sequence (Table 1, Figure 1). Thus, we conclude that MD simulations of the ground state are a sound starting point for the excited-state characterization. Experimentally, the spectral uniformity of the ground states is broken upon photoexcitation of B. wQT and EGFP display monoexponential fluorescence decays with close lifetimes and quantum yields, thereby indicating very similar excited-states and radiative rates. At odds, wQ reveals a faster bi-exponential decay associated with two distinguishable spectra. MD simulations at the excited state readily account for these differences. Under the reasonable hypothesis of fluorescence quenching whenever the chromophore structure is intramolecularly twisted to a large extent (angles φ,τ=±90, Figure 5), wQ is much more amenable to spontaneously reach the twisted configuration than the other two mutants, which are less prone to intramolecular torsion of the chromophore, possibly on account of the hindrance of the T65 residue. Note that MD predicts well the trend of non-radiative lifetimes of the three proteins. 19 ACS Paragon Plus Environment

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Yet wQ and wQT are readily switchable to trans protonated state At, wherefrom they are reconverted to B by photoexcitation at shorter wavelengths (405 nm) or by pH-dependent thermal recovery. The photoswitching properties of wQ and wQT are rather unlike (wQ switches faster and is much more resistant to fatigue), but nonetheless strikingly opposite to the nonphotoconvertible behavior of EGFP. These results suggest poor correlation between the emitting behavior of the excited state and the switching capability of the protein. Yet MD simulations at the excited state propose a solution to this intriguing scenario. Whenever p-HBI is strongly twisted around the τ-dihedral angle that oversees the cis-trans isomerization, the NH2 of Q222 prevalently adopts a conformation that establishes two H bonds one to S205 and the other to the cN2 of the chromophore imidazolidinone. In the planar chromophore conformation, cN2 is almost never contacted by Q222 because of steric hindrance by S/T65 and by the chromophore phenolate. When τ increases towards 90 the imidazolidinone ring swinging brings the cN2 closer to Q222 NH2, allowing an additional hydrogen bond to be formed. The emerging hypothesis is thus that the presence of Q222 selectively stabilizes twisted chromophore conformations, eventually leading to cis-trans photoisomerization. We should nonetheless point out that just the energetic stabilization (and over-representation in the population) of the twisted conformations could not be the sufficient reason for photoswitching. In fact, off-photoswitching quantum yields of wQ and wQT differ by just a factor of 3 (Table 2), whereas MD simulations indicate that the global fraction of doubly Hbonded twisted conformations of wQ are almost tenfold the wQT counterpart. Even considering the inevitable artificial scenario given by the simulations, such a difference cannot be ignored, also because the large difference in the chromophore twisted conformations accounts well for the observed lifetime. More likely, the peculiar doubly H-bonded conformation of Q222-NH2 20 ACS Paragon Plus Environment


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modulates the electronic density on excited p-HBI, leading, although infrequently, to a conical intersection and the trans configuration. This effect could not be revealed by our MD simulations, whose use of static partial charges for the excited state neglects both polarization by the surrounding protein and water environment and, in particular, electron density relocation. Notably, Morozov et al.38 suggested that, for a hula-twist mechanism, the negative charge moves mostly to the imidazolidinone ring close to the conical intersection. In Q222 mutants the H-bond between NH2-Q222 and cN2 might favor this relocation process and, thereby, promote the isomerization. Our Q-RSFPs have not only value for elucidating the photoswitching mechanism. On account of its fast switching, good contrast ratio and striking resistance to photoswitching fatigue under strong illumination conditions, we here demonstrate that wQ can be proficiently applied to qOLID imaging to distinguish two protein labels with the same color color.


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CONCLUSIONS The dynamic behavior of fluorescence in RSFPs addresses the peculiar requisites of superresolution techniques. To date, most RSFPs have been engineered by a rational approach only to a limited extent, and the correlation between the mutation pattern and the photophysical switching property is largely anecdotic. Yet we recently introduced the E222Q replacement as a mutation capable to restore the intrinsic cis-trans photoisomerization properties of the chromophore in otherwise non-switchable Aequorea proteins. A full understanding of the relationship between Q222 and photoswitching is expected to have a strong impact on the rational development of new and improved RSFPs (Q-RSFPs if they contain Q222). In this study, we tackled this issue by a mixed experimental and theoretical approach making use of two simple Q-RSFPs variants (wQ and wQT) and their ancestor, the popular green mutant EGFP. All findings converge on one point: the switching behavior must be a property strictly related on the sole action of Q222 on the excited state of the chromophore. Molecular dynamics simulations afford a simple yet elegant scenario: largely twisted chromophore structures around the double bond (including hula-twist configurations) are uniquely stabilized by Q222 via Hbonds. Likely, these H-bonds subtly modulate the electronic properties of the chromophore, enabling the conical intersection that connects the excited cis to ground trans chromophore. Our study substantiates E222Q as one of those single mutations that, by means of an elusive structural modification, change dramatically the functional phenotype of a protein. The relevance of this finding is witnessed by the capability to distinguish quantitatively wQ from the spectrally identical EGFP by a simple imaging approach (qOLID), which makes no use of filter or spectral deconvolution. This example shows how our rational approach to photoswitching is a powerful



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and time-saving way to enable novel imaging strategies that expand the range of applications of genetically encodable fluorescent probes.


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MATERIALS AND METHODS Protein construction and expression. The recombinant E2GFP was obtained by cloning E2GFP in pET28c between NdeI and HindIII, taking advantage of the 6Htag at the N-terminal of the vector. Recombinant EGFP, wQ and wQT were obtained by performing single point mutations onto the sequence of EYQ1 in p37plus IBA vector.22 In particular wQ was generated by using the primer Y203T: 5’-CAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAA-3’ and wQT by using Y203T and S65T primers: 5’-GTGACCACCCTGACCTACGGCGTGCAGTGCTTC-3’. EGFP was obtained by reverting the mutation on 222 amino acid of wQT by using the primer Q222E: 5’ CGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGG-3’. Recombinant GFP mutants were transformed in E. coli BL21(DE3) strain (Invitrogen) and the cells were grown at 37°C to an absorbance at 600nm of 0.6. Protein expression was induced with 250µM isopropyl-β-D-thiogalactoside (IPTG - Sigma) for 16h at 28°C or with 200µg/L anhydrotetracycline hydrochloride (AHT - IBA) for 30h at 30°C for pET28c and pASKIBA37Plus vectors respectively. Cells were harvested by centrifugation (4500g, 20min, 4°C) and frozen at -20°C. Cells were then resuspended in ice cold lysis bufferA (50mM tris-HCl pH 8.0, 150mM NaCl, EDTA-free protease inhibitor cocktail (Roche)) and lysed by sonication on ice followed by 1h treatment with 0.1% Triton-X100 at 4°C. After removal of the debris by centrifugation (12000g, 1h, 4°C), the supernatant was mixed with 5mL of NiNTA Agarose beads (QIAGEN) and incubated on a rotor for 4h at 4°C. The His - tagged protein was then eluted in bufferA + 500mM Imidazole. The eluted protein was exchange in bufferB (20mM diethanolamine (DEA) pH 8.5). For X-Ray crystallography, two additional purification steps were carried out in a fast protein liquid chromatography system - AKTAxpress (GE Healthcare). The protein was purified by anion exchange (QTrap, GE Healthcare) using a linear gradient with



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10mL column-volumes duration from 0 to 250mM Na2SO4 in DEA 20mM pH8.5. A sizeexclusion chromatography with Superdex200 10/300GL (GE Healthcare) pre-equilibrated with buffer C (20mM DEA pH8.5, 70mM Na2SO4) was used as final purification step. Protein purity and monomeric state was finally evaluated by SDS-PAGE and the concentration was determined by UV absorption measurements. Constructs, Cell Culture and Transfections The mammalian expression vector encoding for wQ-GPI was generated by site directing mutagenesis of GFP-GPI construct41 using the primer: E222Q: 5’-CACATGGTCCTGCTGCAGTTCGTGACC-GCCGCC-3’. The construct COX8EGFP is a kindly gift of Prof. A. S. Verkman.42 CHO K1 cells were provided by American Type Culture Collection (CCL-61 ATCC) and grown in Dulbecco’s modified Eagle medium F-12 nutrient mix (D-MEM/F-12, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). For live imaging, 12×104 cells were plated 24 hours before transfection onto a 35-mm glass-bottom dish (WillCo-dish GWSt-3522). Cells were imaged 24 hours after transfection. Transfections of all constructs were carried out using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. In all experiments, cells were maintained at 37°C in a 5% CO2 atmosphere. Steady-state absorption and fluorescence spectra. Absorption and fluorescence spectra were recorded in cuvettes with 1 cm optical path (Hellma, Müllheim, Germany) at 23 ºC on a Jasco V550 spectrophotometer (Jasco, Easton, MD, USA) and a Cary Eclipse spectrofluorometer (Varian, Palo Alto, CA, USA), respectively. Extinction coefficient and quantum yield determination. The concentration of purified protein was determined from its absorption at 280 nm in the folded state using the extinction coefficient predicted from the protein sequence according to the Pace’s method.44 The degree of


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chromophore maturation was calculated from the protein concentration by the Ward’s method, which requires protein denaturation in 0.2 M NaOH and absorption read-out at 448 nm: assuming 100% maturation, ε448 = 44,200 M-1cm-1.45, 46 Protein quantum yields were determined by using Fluorescein as standard (ΦFluo=0.92 in NaOH 0.1 M). More in detail, the absorption and fluorescence emission spectra (λex=450 nm) of a protein solution (pH = 8.2, DEA 20 mM) and fluorescein (NaOH 0.1 M) were collected sequentially; the absorption of both samples was kept below 0.08 to avoid inner filter effects. The protein quantum yield was calculated by the equation: Φ P = Φ Fluo

FP AFluo AP FFluo

[2]

where FP and FFluo are the integrated fluorescence intensities from 460 to 700 nm, AP and AFluo are the absorbances at 450 nm. Time-resolved fluorescence measurements (TCSPC) Time-correlated single-photon counting measurements of fluorescence time-decays were recorded by means of a confocal scanning laser microscope Leica TCS SP5 (Leica Microsystems, Mannheim, Germany) operating in Fluorescence Lifetime Imaging mode (FLIM). Fluorescence was observed by collecting the emission in a given wavelength range (set by the spectral system of the Leica Confocal microscope) by a photomultiplier tube interfaced with a Time Correlated Single Photon Counting card and setup (PicoHarp 300, PicoQuant, Berlin); FLIM acquisitions lasted until 100 photons were collected on average in each pixel. Protein solutions were placed in the same cuvettes used for steady-state spectroscopy and imaged by a 20× 0.6 NA air objective (Leica Microsystems, Berlin, Germany). Line scanning speed was set to 400 Hz. The pinhole aperture was set to 1.0 Airy. Lifetime data were analyzed by SymphoTime software (PicoQuant, Berlin).



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For global lifetime determination, fluorescence was collected in the 500-550 nm range. Instead, for decay-associated spectra (DAS), fluorescence decays were sequentially detected in 5 nm intervals over the wavelength range 480-610 nm. Then, the decay curves collected at multiple emission wavelengths were simultaneously analyzed by the global procedure with linking lifetimes, using a steady-state fluorescence emission spectrum to normalize decay areas. This procedure, reported in ref. 47 yielded final DAS. In all cases, protein solutions were kept at pH 8.2 (20 mM diethanolamine buffer). Protein photoswitching in solution. Proteins were dissolved in buffers (acidic/neutral buffer: 2 mM citrate/10 mM phosphate; basic buffer: diethanolamine 20 mM) at selected pH in 1-cm path quartz cuvettes (Hellma, Müllheim, Germany) supplied with a magnetic stirrer. Under stirring, the protein solution was front-face irradiated for selected times by using continuous wave laser light (405 nm: FP-40/7AF-AV-SD5 laser, Optoprim, Monza, Italy; 488: Stabilite 2017 ion laser, Spectra-Physics, Mountain View, CA, USA). The power of the laser beam exiting from the solution was monitored over time by a Power/Energy Meter 841-PE (Coherent Italia, Milano, Italy). Time-dependent absorption measurements of protein photoswitching (including flash photolysis experiments) were carried out by a Olis RSM 1000 (Olis, Bogart, GA, USA) fast spectrophotometer/spectrofluorometer, collecting a single absorption spectrum in the range 370540 nm every 500 ms. Further description of the flash photolysis measurements is reported in Supporting Information. The absorption spectrum of the At state was determined by performing simple algebra on the spectrum before the photoconversion (S0) and at the photostationary obtained upon prolonged bleaching illumination (S∞): A t = (S∞ − χ ⋅ S0 ) (1− χ )

[3]


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where χ corresponds to the fraction of residual B state at photosteady state. Practically, the value of χ was decreased at step of 0.01 in eq. 3 from 1 to the lowest value that avoided negative peaks in the At spectrum. Protein photoswitching in gel. Polyacrylamide gels embedding fluorescent proteins were prepared according to the following recipe. First, 24.5 µl solution of about 100 µM protein (Tris buffer, pH 7.5) were thoroughly mixed to 17.5 µl of buffer (Tris, pH 7.5), 30 µl of Rotiphorese® Gel 30 acrylamide/bis-acrylamide (Carl Roth, Germany) and 0.75 µl of 10% Ammonium Persulfate (ThermoFisher, USA) in PBS buffer. To this solution was added 1 µl of Tetramethylethylenediamine (ThermoFisher, USA) and 10 µl were transferred to a microscope slide and covered with a coverslip, after 15 min the polymerization was complete and the sample was measured immediately. Protein photoswitching in gel was measured in a custom built confocal (RESOLFT) point scanning system. The setup was built around a Leica DMI 6000 microscope stand equipped with a 100x STEDwhite oil objective lens NA 1.4 (Leica, Wetzlar, Germany) and 2 separate controllable light sources, one for on-switching (405 nm) and the other for off-switching/read-out (488 nm) Gaussian shaped beam. In this study we used diode lasers from Cobolt AB (06-MLD, Cobolt AB, Solna, Sweden), which have the advantage of being directly modulable. The lasers were combined in the same beam using a 458 LP filter (ZT458rdc, Chroma, VT, USA). A Yanus scan system (TILL Photonics, Gräfelfing, Germany) was used for the xy scanning. The stage was a custom-built stage with a built-in z-piezo (PI, Karlsruhe, Germany). The stage was mounted directly onto the objective lens in order to minimize any drift or vibration. The emission light was separated from the laser light using a 488 LP filter (ZT488rdc, Chroma, VT, USA) and further passed a 535 BP filter (E535/70M, Chroma, VT, USA) before being collected by a SPAD 28 ACS Paragon Plus Environment


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(MPD, Bolzano, Italy). Imspector (Abberior, Germany) was used to collect the data and control the hardware. A typical illumination sequence comprised: 7.8 ms dark followed by a 50 s onswitching (405 nm), a 2 ms without illumination (dark) and a 20 ms of off-switching (488); this sequence was cycled 500 times. X-ray Crystallography. The purified protein was concentrated to 20 mg/mL with centrifugal filter devices (Millipore). Crystals were grown using the hanging-drop vapor-diffusion method from a 1:1 mixture of sample and reservoir buffer containing [18% (w/v) PEG3350, 100mM NH4 acetate pH 5.0, and 0.2M NH4F], with the help of seeding technique. Crystals were cryoprotected in mother liquor containing glycerol 25% (w/v) and subsequently flash-cooled in liquid nitrogen before data collection at cryogenic temperature (100 K) at beam line XRD1-ELETTRA (Trieste, Italy). The structure was solved by molecular replacement using the structure of the fluorescence protein E2 as a search model (PBD entry code 2O24).48 Structure resolution and refinement were carried out with the CCP4i program package.49 Refinement statistics can be found in Table S1.1 (SI1). The crystal structure of wQ and structure factors have been deposited in the Protein Data Bank with entry codes 6FLL. Molecular dynamics simulations All-atom force-field based MD simulations were performed on the three mutants (EGFP, wQ and wQT) in explicit solvent using the amber99SB*-ILDN force field.50, 51 We performed Hamiltonian replica exchange (HREX) simulations to sample the (ground-state) conformation of each mutant. From these simulations we extracted the starting structures for 500ps long MD simulations using a force field modeling the excited-state chromophore, taken from ref.

34

. In brief, the potential in

34

reproduces the excited-state two-

dimensional potential energy surface for the torsion around the two dihedral angles in the chromophore methine bridge, calculated by high-level ab-initio quantum mechanics methods for


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the chromophore in the gas phase. The additional details of MD simulations methods are reported in the Supporting Information.

Cell imaging and switching sequences Cell fluorescence was measured using a Leica TCS SP5 SMD inverted confocal microscope (Leica Microsystems AG). Glass-bottom Petri dishes containing fixed cells were mounted in a thermostated chamber at 37°C (Leica Microsystems, Wetzlar, Germany) and viewed with a 100x 1.3 NA oil-immersion objective (Leica Microsystems). The pinhole aperture was set to 1.0 Airy. Excitation was provided by an Ar+ laser source (488 or 514 nm) and/or by a solid-state pulsed diode laser emitting at 405 or 470 nm (Picoquant, Berlin, Germany). A typical photoswitching sequence was carried out by using the Live Data Mode Wizard of the LAS Software alternating a single illumination step at 405 nm to switch-on the GPI-wQ (power at the objective: 50µW) and six 488 nm pulses (power at the objective: 40µW) to switch-off the protein and to collect the fluorescence. The 488 nm wavelength excites also the COX8-EGFP. The emission was collected in the spectral range 500-540nm. qOLID analyses were carried out with a custom-made ImageJ (http://imagej.nih.gov/) plugin available upon request.



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33. Arpino, J. A. J., Rizkallah, P. J., and Jones, D. D. (2012) Crystal Structure of Enhanced Green Fluorescent Protein to 1.35 angstrom Resolution Reveals Alternative Conformations for Glu222, PLoS ONE 7, 8. 34. Jonasson, G., Teuler, J. M., Vallverdu, G., Merola, F., Ridard, J., Levy, B., and Demachy, I. (2011) Excited State Dynamics of the Green Fluorescent Protein on the Nanosecond Time Scale, J Chem Theory Comput 7, 1990-1997. 35. Coquelle, N., Sliwa, M., Woodhouse, J., Schirò, G., Adam, V., Aquila, A., Barends, T. R. M., Boutet, S., Byrdin, M., Carbajo, S., De la Mora, E., Doak, R. B., Feliks, M., Fieschi, F., Foucar, L., Guillon, V., Hilpert, M., Hunter, M. S., Jakobs, S., Koglin, J. E., Kovacsova, G., Lane, T. J., Lévy, B., Liang, M., Nass, K., Ridard, J., Robinson, J. S., Roome, C. M., Ruckebusch, C., Seaberg, M., Thepaut, M., Cammarata, M., Demachy, I., Field, M., Shoeman, R. L., Bourgeois, D., Colletier, J.-P., Schlichting, I., and Weik, M. (2017) Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography, Nat Chem 10, 31. 36. Martin, M. E., Negri, F., and Olivucci, M. (2004) Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution, J Am Chem Soc 126, 5452-5464. 37. Zhang, Q. Q., Chen, X. B., Cui, G. L., Fang, W. H., and Thiel, W. (2014) Concerted Asynchronous Hula-Twist Photoisomerization in the S65T/H148D Mutant of Green Fluorescent Protein, Angew Chem Int Edit 53, 8649-8653. 38. Morozov, D., and Groenhof, G. (2016) Hydrogen Bond Fluctuations Control Photochromism in a Reversibly Photo-Switchable Fluorescent Protein, Angew Chem Int Edit 55, 576-578. 39. Mao, S., Benninger, R. K., Yan, Y., Petchprayoon, C., Jackson, D., Easley, C. J., Piston, D. W., and Marriott, G. (2008) Optical lock-in detection of FRET using synthetic and genetically encoded optical switches, Biophys J 94, 4515-4524. 40. Petchprayoon, C., Yan, Y., Mao, S., and Marriott, G. (2011) Rational design, synthesis, and characterization of highly fluorescent optical switches for high-contrast optical lock-in detection (OLID) imaging microscopy in living cells, Bioorg Med Chem 19, 1030-1040. 41. Lenne, P. F., Wawrezinieck, L., Conchonaud, F., Wurtz, O., Boned, A., Guo, X. J., Rigneault, H., He, H. T., and Marguet, D. (2006) Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork, Embo J 25, 32453256. 42. Kneen, M., Farinas, J., Li, Y., and Verkman, A. S. (1998) Green fluorescent protein as a noninvasive intracellular pH indicator, Biophys J 74, 1591-1599. 43. Battisti, A., Digman, M. A., Gratton, E., Storti, B., Beltram, F., and Bizzarri, R. (2012) Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging, Chem Comm 48, 5127-5129. 44. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein, Protein Sci 4, 2411-2423. 45. Sniegowski, J. A., Lappe, J. W., Patel, H. N., Huffman, H. A., and Wachter, R. M. (2005) Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein, J Biol Chem 280, 26248-26255. 46. Sniegowski, J. A., Phail, M. E., and Wachter, R. M. (2005) Maturation efficiency, trypsin sensitivity, and optical properties of Arg96, Glu222, and Gly67 variants of green fluorescent protein, Biochem Biophys Res Commun 332, 657-663.


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FIGURE 1

Figure 1. Absorption and fluorescence properties. (a) Absorption of wQ at pH 8.2 (blue) and pH 5.0 (red); fluorescence emission of wQ by excitation at 450 nm (black). (b) Lifetime decays of wQ (green) and wQT (black) at pH 8.2. (c) Emission spectra of wQ associated to the two lifetime components: τ1 (blue), τ2 (red) at pH 8.2; the dotted black line corresponds to steady state emission at the same excitation wavelength, 470 nm.


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FIGURE 2

Figure 2. Reversible photoswitching properties of wQT. (a) Off-photoswitching: difference absorption spectra at different times upon illumination at 488 nm (0.3 W/cm2). (b) Onphotoswitching: difference absorption spectra at different times upon illumination at 405 nm (3 mW/cm2) of the photoconverted form. (c) Comparison between the molar absorption of B state 36 ACS Paragon Plus Environment


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(blue line), the photosteady state (dashed green line), and the At state (red line). (d) Thermal recovery: difference absorption spectra at different times under dark at pH 7. (e) Thermal recovery kinetics at pH 6.7 (red line) and 7.1 (black line). (f) Thermal recovery: kinetic rate constant k vs. pH fitted to a single-site ionization titration curve (eq.1).


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FIGURE 3

Figure 3. Photoswitching properties at high bleaching (488 nm) and reactivation (405 nm) illumination intensities. (a) Photoswitching cycles of wQ by alternating bleaching and reactivation illuminations; the colored scheme on top represents the illumination sequence (see Materials and Methods for details). (b) Average (500 cycles) off-switching kinetic curves of wQ at different bleaching powers: 0.88 kW/cm2 (red), 3.5 kW/cm2 (green), 10.6 kW/cm2 (cyan), 42.4 kW/cm2 (blue), and 41.3 kW/cm2 reactivation power (pulse of 50 µs); inset: comparison between wQ (blue) and wQT (black) at constant 42.4 kW/cm2 bleaching power, using log-scale for time to highlight differences between the off-switching rates. (c) Decrease in initial fluorescence along the switching cycles (fatigue) of wQ (blue), wQT (black), and rsEGFP2 (red) at constant 42.4 kW/cm2 bleaching power.



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FIGURE 4

Figure 4. Structure of wQ. (a) Ribbon representation of wQ (gray) with the internal chromophore (carbon atoms colored in green). (b) Local environment of the chromophore, consisting of a cyclized tripeptide made of S65, Y66 and G67, highlighting the presence of water molecules (red spheres) and hydrogen bonding interactions (black dashed lines). Labeled amino acids are in the one-letter code. The electron density overlaid on the chromophore and neighboring solvent molecules is also shown at a 1.5σ contour level (2fofc map, colored in blue). The alternative conformation of Ser65 (carbon atoms colored in yellow) partially interrupts the interactions involving V61, S65 and Q222. The sphere colored in cyan indicates the position of the water molecule present in the superimposed coordinates of the EGFP structure (PDB code: 2y0g), which mediates the hydrogen bond network in the vicinity of the chromophore. As shown by the electron density map, this water molecule is absent in the crystal structure of wQ.


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FIGURE 5

Figure 5. a) Percentage of 500ps MDEX simulations leading to φ or τ twisting event (solid black), τ twisting event (shaded black and red), τ twisting event accompanied by 2Hb-Q222 conformation (shaded red). b) Instantaneous values of φ and τ during all MDEX simulations of wQ. The values for all snapshots are shown in gray and those with 2Hb-Q222 in red. The curves in the bottom graph show the percentage of snapshots containing 2Hb-Q222 as a function of τ for wQ, wQT and wQQ→E (see text). c) Ground-state (left) and excited-state (right) representative MD snapshots of wQ.



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FIGURE 6

Figure 6. qOLID images of GPI-wQ and COX8-EGFP expressed in CHO cells. a) Average stack intensity; b) map of photoswitchable fraction (fSW), corresponding to GPI-wQ; c) map of nonphotoswitchable fraction (fNS), corresponding to COX8-EGFP; d) merge of fSW (red) and fNS green, highlighting the two intracellular localizations. Scale bar (panel a): 10 µm


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SCHEME 1 Scheme 1. Cis-trans photoisomerization of p-HBI chromophore in Q-RSFPs. O N N

O

hν (488 nm)

B bright

hν (405 nm)

HO O N N At dark



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Table 1. pKa and absorption and fluorescence features of the fluorescent proteins Absorption

Fluorescence

Cis

a

Trans

Cis

Protein

pKa

λB (nm)

εB (M cm-1)

λA’ (nm)

εA’ (M cm-1)

λAt (nm)

εAt (M cm-1)

λB (nm)

Φ

τ1 (ns)

τ2 (ns)

τNRa (ns)

wQ

6.1

476

57400

390

28800

403

28600

504

0.45

2.26 (53.8 %)

1.31 (46.2 %)

3.3

wQT

5.7

482

64300

392

27100

405

27600

505

0.71

2.56

-

8.8

EGFP

5.9

488

60000

396

28700

-

-

508

0.73

2.61

-

9.7

-1

-1

-1

Non-radiative lifetime as calculated from the amplitude-average lifetime and quantum yield.

Table 2. Photoswitching features of wQ, wQT, and other RSFPs.

Mutant

At/Ba (% / %)

D

wQ

81 / 19

wQT

76 / 24

k0 (s )

k∞ (s-1)

4.5

∼10-4

3.2

∼10

-1

-3

pKr

ϕoff ⋅103

ϕon ⋅103

(20.6±0.8)⋅10-2

8.92±0.05

2.7±0.1

70±10

-2

9.62±0.01

1.06±0.01

25±3

4.7

26

0.16

165

3.2

150

(59.2±0.1)⋅10

Mut2Q22 Dronpa IrisFP

52

53 a

Composition of photostationary state at pH 7 under 488 nm as obtained from the absorption spectrum


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ASSOCIATED CONTENT Supporting Information Supporting information. Additional steady-state spectra, flash-photolysis measurements, qOLID image, computational details, and analysis of MD simulations (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected] ; *[email protected] *[email protected] Author Contributions B.S. R.N. and R.B. designed research; B.S., E.M., G.A., J.D., G.D., I.T., G.G., R.N., and R.B. performed research; B.S., G.A., J.D., G.G., R.N., and R.B. analyzed data; all the authors wrote the paper. Funding Sources This research, in the framework of the project “DIAMANTE – Diagnostica Molecolare Innovativa per la scelta terapeutica personalizzata dell’adenocarcinoma pancreatico” (grant number CUP I56D15000310005), was supported by the Regione Toscana Bando FAS Salute 2014 (Italy). B.S. acknowledges the funding from the Short Term Mobility 2015/2016 of the National Research Council of Italy (CNR). I.T. and J.D. acknowledge the funding from the ERC 44 ACS Paragon Plus Environment


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starting grant (http://dx.doi.org/10.13039/501100000781). J.D. salary was funded by the Carlsberg Foundation.

ACKNOWLEDGMENT The authors gratefully acknowledge C. Di Rienzo (Scuola Normale Superiore, Pisa Italy), C. Viappiani (Department of Physics, University of Parma, Italy), C. Filippi (Faculty of Science and Technology, Twente University) and F. Pennacchietti (Department of Applied Physics and Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Sweden) for useful discussions. ABBREVIATIONS RSFP, reversibly switchable fluorescent proteins; HREX, Hamiltonian Replica Exchange; qOLID, quantitative Lock-In Detection


Role of Gln222 in Photoswitching of Aequorea Fluorescent Proteins: A

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