# Light-Induced Conformational Heterogeneity Induces Positive Photoswitching in Photoconvertible Fluorescent Proteins of the EosFP Family

Jip Wulffelé, Arijit Maity, Isabel Ayala, Serge Gambarelli, Bernhard

Brutscher, Dominique Bourgeois

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## ► To cite this version:

Jip Wulffelé, Arijit Maity, Isabel Ayala, Serge Gambarelli, Bernhard Brutscher, et al., Light-Induced Conformational Heterogeneity Induces Positive Photoswitching in Photoconvertible Fluorescent Proteins of the EosFP Family. Journal of the American Chemical Society, 2025, 147 (12), pp.10357-10368. 10.1021/jacs.4c17311 . hal-05001616

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# Light-induced conformational heterogeneity induces positive photoswitching in photoconvertible fluorescent proteins of the EosFP family

Jip Wulffelé<fnref n="1" />ᵃ, Arijit Maity<fnref n="1" />ᵃ, Isabel Ayala<fnref n="1" />, Serge Gambarelli<fnref n="2" />, Bernhard Brutscher<fnref n="1" />*, Dominique Bourgeois<fnref n="1" />*

**ABSTRACT:** Green-to-red photoconvertible fluorescent proteins (PCFPs) of the EosFP family are commonly used in ensemble pulse-chase and single-molecule localization or tracking approaches. However, these fluorescent proteins exhibit highly complex photophysical behaviors. In the green-form, recent NMR experiments revealed that mEos4b and other PCFP variants exist in two different conformational states at thermal equilibrium, which limits their effective photoconversion efficiency. Here, we investigate the conformational heterogeneity of mEos4b in the photoconverted red-form, employing a combination of solution NMR, UV-vis spectroscopy and fluorescence imaging. Only a single red population of mEos4b is observed at thermal equilibrium. However, a second population emerges under illumination with 405 nm or 488 nm light, which slowly decays in the dark or can be swiftly reverted under 561 nm light. This second population manifests itself through a pH-dependent positive photoswitching mechanism that adds to the already characterized negative photoswitching assigned to *cis-trans* isomerization of the chromophore. Our data indicate that positive photoswitching, instead, results from the light-induced formation of a second fluorescent state with a *cis* configuration of the chromophore that exhibits a substantially increased pKa. Such a mechanism, suggested to result from rewiring of the H-bonding network around the first amino acid of the chromophore, adds to the panoply of switching scenarios observed in fluorescent proteins. It bears consequences for the spectroscopic characterization of PCFPs, reduces their apparent brightness and generates short-lived off times perturbing single-molecule localization microscopy applications.

## INTRODUCTION

Photoconvertible fluorescent proteins (PCFPs) have remained attractive genetically-encoded cellular markers for advanced fluorescence microscopy approaches over recent years.<fnref n="1" />,<fnref n="2" /> Members of the EosFP family³ such as mEos3.2,⁴ mEos4b,⁵ pcStar⁶ or mEosEM⁷ are particularly popular because of their relatively high photon budget, good monomeric quality, and, in the case of mEos4b and mEosEM, strong fixation resistance. Taking advantage of the ideal labeling specificity offered by genetically encoded markers, these PCFPs have been repeatedly used for quantitative counting⁸⁻¹⁰ or tracking¹¹⁻¹⁴ experiments at the single molecule level. Green-to-red PCFPs are initially in a green-emitting form and upon absorption of a violet photon (~405 nm) stochastically photoconvert to a red-emitting form which is detected upon excitation with yellow-green light (~561 nm). They all possess a histidine at the first position of the chromophore triad (His 62, mEos4b sequence numbering). Photoconversion proceeds through polypeptide backbone cleavage between His 62 and Phe 61, which elongates electron conjugation to the imidazole ring of His 62. Despite various experimental and computational investigations,³ the underlying mechanism remains incompletely understood. In turn, in addition to photoconversion, multiple blinking and bleaching behaviors have been discovered in recent studies,³,¹⁵⁻¹⁹ revealing a high level of complexity of the photophysics of EosFP variants in both their

green- and red-forms (Figure 1, a more detailed version is provided in Figure S1).

At the single molecule level, blinking in EosFP-derived PCFPs has been shown to involve both short-lived and long-lived non-fluorescent (dark) states. While short-lived dark states have been assigned to radicals,²⁰,²¹ as also observed in other FPs,²²⁻²⁵ long-lived dark states have been shown to result from *cis-trans* isomerization of the chromophore, a hallmark of many reversibly photoswitchable FP (RSFP) variants such as Dronpa or rsEGFP2.²⁶,²⁷ This type of photoswitching is said to be “negative”, ie the light-exciting fluorescence (~561 nm) promotes switching to a nonfluorescent state, while violet light (~405 nm) repopulates the fluorescence state. Intriguingly, an additional “positive” switching behavior has been recently observed at the ensemble level in the red-form of mEos3.2 in yeast²⁸ and in LEA,²⁹ although consequences for potential blinking at the single-molecule level have not been assessed. In such a mechanism, the light exciting fluorescence promotes switching to the fluorescent state, while violet light populates the nonfluorescent state. Overall, the complex blinking behavior of EosFP variants complicates single-molecule imaging experiments, generating overcounting in PALM,³⁰,³¹ track interruptions in sptPALM¹⁵ and possibly mislocalization in Minflux.³² In addition, different routes for photobleaching have been described in those PCFPs,

<footnote n="1">¹ Université Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 71 avenue des Martyrs, 38044 Grenoble Cedex 9, France</footnote>

<footnote n="2">² Université Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, Grenoble F-38000, France</footnote>

<footnote>ᵃThese authors contributed equally to this work</footnote>
depending on illumination wavelength and light power den-
sity.<sup>19,33,34</sup> A detailed understanding of the photophysics of
PCFPs is thus of key importance to avoid misinterpretation of
microscopy data,<sup>30,35</sup> guide rational engineering of better behav-
ing variants<sup>6,34,36</sup> and optimize illumination schemes in ad-
vanced imaging.<sup>15,37</sup>

<figure><img src="image_3.png" /><figcaption>Figure 1. Simplified photophysical model of EosFP-like PCFPs.</figcaption></figure>

Recently, solution NMR studies introduced yet another layer of
complexity in the photophysics of PCFPs.<sup>18</sup> Two conforma-
tional states (named the A and B states) were observed to coex-
ist in the green-form of mEos4b, populated about equally at
thermal equilibrium and interconverting on the minutes time-
scale in the dark at 35°C. The two states were assigned to dif-
ferent H-bonding networks involving His62 of the chromo-
phore, as well as the neighboring amino acid tetrad Glu144-
His194-Glu212-Gln38. The equilibrium between the two states
can be shifted towards the B state by illumination with 405 nm
light. In addition, while only the A state efficiently photocon-
verts to the red- form, the B state appears more prone to photo-
bleaching.<sup>18</sup> While this study indicated that conformational het-
erogeneity in the green-form of EosFP variants plays an im-
portant role in limiting photoconversion efficiency, it remained
to be established whether similar conformational heterogeneity
also exists in their red-form (Figure 1).

In the present work, we employ a combination of NMR, UV-
vis spectroscopy and fluorescence imaging to investigate the
red-form of mEos4b. Strikingly, in contrast to the green-form,
the red-form populates only a single conformational state at
thermal equilibrium. However, under illumination with violet
or cyan light, conformational heterogeneity is elicited again,
giving rise to a positive photoswitching behavior similar to the
one previously noticed in mEos3.2<sup>28</sup> and LEA.<sup>29</sup> We study the
possible mechanisms at play and conclude that positive pho-
toswitching in mEos4b and other EosFP variants originates
from the coexistence of two fluorescent states displaying differ-
ent chromophore pKa's and thus widely different effective
brightness depending on pH. We show that positive switching
in mEos4b generates short-lived off-times in single-molecule
imaging experiments, notably at low pH. Overall, this work
highlights a previously overlooked switching mechanism at
play in photoconvertible fluorescent proteins, driven by light-
induced conformational heterogeneity.

RESULTS

The red-form of mEos4b is homogeneous in solution

We recently reported that the green-form of mEos4b is hetero-
geneous with almost equal percentage of two co-existing con-
formational states (A and B).<sup>18</sup> Inspection of the <sup>1</sup>H-<sup>15</sup>N NMR

spectrum of a mEos4b sample freshly photoconverted with 405
nm light revealed minor peaks flanking major peaks for a num-
ber of residues, suggesting that the red-form of mEos4b may
also be heterogeneous (Figure 2A). However, leaving the sam-
ple in the dark for several hours resulted in a progressive disap-
pearance of these minor peaks, while at the same time the in-
tensities of the main peaks increased. At thermal equilibrium,
red mEos4b is present in a single conformation, as can be ap-
preciated from the <sup>1</sup>H-<sup>15</sup>N NMR spectrum shown in Figure S2,
with all correlation peaks sequentially assigned to a single spe-
cies. The thermal relaxation kinetics could be fitted to a mono-
exponential function with a time constant of 160 min at 35 °C
(Figure 2B).

As the green state heterogeneity results from a difference in
side-chain protonation of His62 and Glu212, we also investi-
gated the protonation state of these two residues in the red-form
of mEos4b by NMR. First, chemical shift assignments of all <sup>1</sup>H-
<sup>13</sup>C and <sup>1</sup>H-<sup>15</sup>N moieties of the red chromophore were obtained
(Figure S3A and S3B, Table S1). The histidine <sup>13</sup>CD<sub>2</sub> chemi-
cal shift is particularly sensitive to the tautomeric state of the
imidazole. The value of 135 ppm measured for His62 indicates
that the imidazole side chain is protonated at the ND<sub>1</sub> position.
This result is further confirmed by NOE-based inter-proton dis-
tance measurements (Figure S3C). It is also in agreement with
NMR data previously reported for peptide fragments of the pho-
toconvertible protein Kaede,<sup>38</sup> harboring the same red chromo-
phore. We also attempted to identify the protonation state of the
fully conserved Glu212 at its carboxylic side chain. Unfortu-
nately, no side-chain chemical shift information could be ob-
tained for this residue, most likely due to conformational ex-
change dynamics on a milliseconds time scale, inducing exten-
sive line broadening of NMR signals.

405-nm light promotes red-form heterogeneity

Application of 405-nm light on a thermally relaxed red sample
regenerated the minor NMR peak species, while decreasing the
intensity of the major peaks (Figure S4), suggesting that popu-
lation heterogeneity can be induced by UV light from the ther-
mally stable red-form. Furthermore, the process could be re-
verted by application of 561 nm light (Figure S4), indicative of
a photo-induced reversible switching mechanism. In the follow-
ing, we will refer to the thermally relaxed red-form as R1, while
the species populated upon 405 nm illumination will be referred
to as R2. About 35 ± 5 % of the red mEos4b molecules are con-
verted to the R2 state at pH 7.5 and 35°C, as derived from the
maximal fractional increase of NMR peak intensities upon ther-
mal relaxation (Figure 2B). Of note, the intensities of the minor
peaks assigned to R2 do not fully compensate for the decrease
in R1 signal, suggesting that R2 is a heterogeneous confor-
mation, most likely with exchange dynamics on the ms time
scale, causing NMR line broadening and loss of NMR signal.

An important consequence of the above-mentioned finding is
that under sole 405 nm illumination, photoconversion to R1 is
incomplete and additional 561 nm, or thermal relaxation, is re-
quired to complete the process (Figure 2C). This suggests that
in a fluorescence microscope, depending on R2 brightness, ef-
fective observation of all photoconverted single molecules may
require both 405 and 561 nm light illumination.
<figure><img src="image_4.png" /><figcaption>**Figure 2.** Experimental observation of an additional 405-induced red state (R2). (A) Changes observed in ¹H-¹⁵N correlation spectrum (selected region) of green mEos4b (left panel), just after illumination with low-power 405 nm light (central panel), and after relaxation in the dark for several hours (right panel). Peaks only visible in the spectrum of freshly photoconverted mEos4b (circles), are assigned to the R2 species. (B) Thermal relaxation kinetics (35°C, pH 7.5) of NMR peaks assigned to R2 (decay) and R1 (buildup). Both data sets were fitted (solid lines) to a 2-state R1-R2 exchange process with a time constant of 160 min, and a R2 population at t=0 of ~35±5%. (C) mEos4b green-to-red photoconversion kinetics (35°C, pH 7.5) measured by ¹H-¹⁵N NMR upon *in-situ* illumination. The light power densities (measured at the sample top) were 4.5 mW/cm² (405 nm) and 500 mW/cm² (561 nm). (D) Concurrent recording of *in situ* ensemble fluorescence (red trace) and NMR (black dots, monoexponential fit shown as black line, inset) signals during R1-R2 conversion induced by 405 nm light (40 mW/cm²). (E) mEos4b molecules immobilized in PAA gel (pH 7.4) were photoconverted using 10 W/cm² 405 nm light and subsequently cycled between fluorescent and non-fluorescent states using 10 W/cm² 561 nm light and 10 W/cm² 405 nm light in an alternating manner. The fluorescence signal was recorded every 50 ms by illumination with 10 W/cm² 561 nm light for 5 ms. Arrows indicate the instances of positive photoswitching. Shown is the mean ± sd of 4 measurements. (F) Superposition of ¹H-¹⁵N NMR spectra (selected region) of the thermally relaxed R1 state (red), 405 nm induced R2 state (violet) and 561-nm induced off state</figcaption></figure>

$(R_{\text{trans}})$ (black) of mEos4b. For additional details see supporting Materials and Methods.

## The 405-induced R2 species is weakly fluorescent or dark at physiological pH.

To assess the fluorescence properties of the R2 state, we monitored ensemble fluorescence emission of mEos4b either directly in the NMR magnet or in a dedicated fluorescence microscope. First, to perform in-situ NMR fluorescence measurements, we developed a home-built fluorimeter setup allowing synchronized fluorescence measurements during NMR experiments (see Figure S5 for the design of the device). Upon 405 nm illumination, the observed R1-to-R2 photoconversion kinetics measured by NMR nicely correlates with the observed fluorescence decay at pH 7.5 (Figure 2D). The observed fluorescence-decrease of about 30% suggests that the R2 state has significantly reduced brightness compared to R1. Note that, due to our experimental setup, we do not expect full quantitative agreement between the measured NMR and fluorescence intensity decrease.

Second, we immobilized mEos4b molecules in a polyacrylamide (PAA) gel matrix at the same pH and monitored ensemble fluorescence using a widefield super-resolution microscope. First, 405 nm light (10 W/cm²) was applied as repetitive 40 ms pulses to promote photoconversion, with (in between) short 5 ms pulses of 561 nm light (10 W/cm²) for red fluorescence readout. During this first illumination period, a mixture of R1 and R2 is formed, as suggested by the NMR data. Then, 561 nm (10 W/cm²) light was applied instead of 405 nm light during the 40 ms pulses. In this second phase, a rapid but transient increase of the ensemble fluorescence was observed, followed by a slower decrease (Figure 2E). The transient increase (positive switching) is attributed to the rapid switching from R2 to R1, suggesting a poorly- or non-fluorescent R2 state consistent with our in-situ NMR results. The subsequent decrease (negative switching) is assigned to the known *cis-trans* isomerization of the red chromophore under prolonged 561 nm illumination,¹⁵ forming a non-fluorescent state which we refer to as *R*<sub>trans</sub>. *R*<sub>trans</sub> is quickly reverted by 405 nm light which forms again the R1-R2 mixture. This behavior could be observed over repeated cycles (Figure 2E), with a progressive loss of fluorescence intensity assigned to photobleaching.

Overall, red mEos4b undergoes both positive and negative reversible photoswitching when excited by either 405 nm or 561 nm light.

In order to demonstrate that the 405 nm induced R2 and the 561 nm induced $R_{\text{trans}}$ states correspond to distinct conformational states, we compared their respective ¹H-¹⁵N NMR spectral fingerprints (Figure 2F). To generate the $R_{\text{trans}}$ state, we illuminated a thermally relaxed red mEos4b sample in the R1 state with 561 nm light to induce the documented *cis-trans* isomerization through negative photoswitching. We observed the appearance of multiple new species (several NMR peaks for a given nuclear site or residue), in line with the “frustrated” highly dynamic $R_{\text{trans}}$ state described previously.¹⁵ A large number of residues show prominent chemical shift changes, as expected for *cis-trans* isomerization.³⁹ While only about 35% of the red mEos4b molecules undergo a conformational change from R1 to R2 upon 405 nm illumination under our experimental conditions, photoswitching by 561 nm light is almost complete (> 95%) (Figure S6A). Clearly, the chemical shifts of the 561-induced ensemble of $R_{\text{trans}}$ states are different from those of the 405 nm induced R2 state (Figure 2F).
<figure><img src="image_5.png" /><figcaption>**Figure 3.** Absorption properties of R2. Photoconverted mEos4b molecules (pH 7.4, universal buffer) were illuminated with ~40 mW/cm² 405 nm light to promote R2 formation for 24 minutes (A) after which the molecules were allowed to thermally relax for ~1 hour (B). Absorption spectra were recorded every second (from red to black and black to red in A and B respectively). C) Difference between the spectrum after illumination with 405 nm light (mixture R1 and R2) and the spectrum after thermal recovery (R1 only). The spectrum of R1 at pH 4 (mostly neutral chromophore) is shown for comparison. R⁻: Red anionic, R⁺: Red neutral, G⁻: Green anionic, G⁺: Green neutral.</figcaption></figure>

## Mechanistic investigations of the nature of R2

To gain more insight into the nature of R2, we first attempted to measure its absorption spectrum using a dedicated spectro-photometer equipped with multiple lasers.⁴⁰ Absorption spectral series of photoconverted mEos4b were first recorded under 405 nm illumination and subsequently in the dark (Figure 3). Illumination with 405 nm light resulted in a decrease of the absorbance bands of the anionic red chromophore (at ~570 nm and ~330 nm), a slight decrease of the absorbance band corresponding to the remaining (not photoconverted) anionic green chromophore (~510 nm) and an absorbance increase at around 450 nm, close to the band of the neutral red chromophore (Figure 3A).¹⁵ In the dark, these changes were essentially reversed, except for the remaining green chromophore that stayed constant (Figure 3B). Thus, the absorbance increase at ~450 nm can be assigned to the formation of R2. The difference spectrum calculated from spectra recorded before and after thermal relaxation (Figure 3C), indicates that absorption of R2 at pH 7.5 is similar to that of R1 at pH 4 in conditions where the chromophore is essentially neutral, with a possible small hypochromic shift. Yet, as R2 is efficiently depopulated by 561 nm light, it must also display residual absorption at this wavelength, close to the anionic band of the R1 red chromophore. Altogether, a possible scenario is that R2, like R1, exists in a neutral and an anionic state with near identical absorption bands, and that excitation of neutral R1 predominantly forms R2, whereas excitation of anionic R2 predominantly forms R1. Consistently, R2 may as well be formed by illuminating R1 with 488 nm light (Figure S7), which decouples photoconversion from R2 formation.¹⁵

Overall, when switching between R1 and R2, the absorption bands that are illuminated grow rather than decay, a quite intriguing behavior.

### Is cis-trans chromophore photo-isomerization involved?

The positive switching behavior observed in red mEos4b is reminiscent of the switching behavior of positive RSFPs such as Padron or rsCherry.⁴¹,⁴² In these RSFPs, off-switching is promoted by excitation of the neutral *cis* chromophore to an anionic (nonfluorescent) *trans* chromophore and on-switching is promoted by excitation of the anionic *trans* chromophore back

to the *cis* configuration. Thus, we wondered whether *cis*-to-*trans* isomerization could also be behind the reversible formation of R2.

We already pointed out, based on NMR ¹H-¹⁵N chemical shifts, that R2 and R<sub>trans</sub> are distinct states, differing either in the chromophore conformation and/or the protein environment (Figure 2F). In order to further investigate whether the chromophore in the R2 state is in a *cis* or *trans* conformation, we recorded ¹H-¹³C correlation spectra, providing a NMR spectral signature of the chromophore, in both the 561 nm induced *trans*-isomerized and the 405 nm induced R2 states (Figure S6B). We could previously show that the ¹³CB chemical shift of the methine bridge carbon is particularly sensitive to *cis-trans* isomerization.³⁹ In the case of the 561-nm induced *trans* state we detected the expected correlation peak indicative of a *trans* chromophore, while no such peak is observed for the 405 nm induced R2 state. Therefore, we conclude that the R1 to R2 transition does not involve a change in the isomeric state of the chromophore, which thus remains in a *cis*- conformation in R2.

### Is radical formation involved?

Next, we checked whether R2 could be a long-lived radical species. Radicals in fluorescent proteins, forming downstream the triplet state, have been reported previously, although most of them are only short-lived.²⁰⁻²² However, as radical(s) in some proteins have been shown to be long-lived and absorb in the near UV,⁴³ the increased absorption at around 450 nm could possibly point to such radical formation. However, the absence of EPR signal of a R2-populated sample strongly suggested that R2 does not consist of a radical species (Figure S8).

### Is protonation involved?

To investigate the influence of protonation/deprotonation events, we probed the pH dependence of R1-R2 switching. Fluorescence data collected on PAA-embedded mEos4b showed that the extent of positive switching increased significantly with decreasing pH (Figure 4A). Additional spectrophotometry experiments performed for an extended pH range from 5 to 9 confirmed this observation, showing that the fractional loss of fluorescence upon 488 nm illumination rapidly reached close to
100% below pH ~5.5 (Figure 4B, Figure S9). The transient increase in absorbance and fluorescence observed at pH 9.1 is likely due to other photophysical processes (possibly residual primed photoconversion<sup>44</sup>) and unrelated to R1-R2 exchange. This pH dependence was also confirmed by NMR measurements. At pH 6.5, the apparent R2 population after 405 nm illumination was significantly increased with respect to the pH 7.5 data (Figure S10). The observation of several ¹H-¹⁵N correlation peaks for a number of residues (Figure S10) also pointed to a more heterogeneous R2 conformational state at low pH, or alternatively a slowdown of the exchange kinetics in the ensemble of R2 states. Finally we also observed that recovery of R1 by 561 nm light was faster at high pH (Figure S11).

The strong pH dependence of the observed state populations and conversion kinetics are in line with the notion that R2 is mostly formed from neutral R1 while R1 is mostly formed from anionic R2. It suggests that positive switching might be a consequence of light-induced proton rewiring in the chromophore environment.

<figure><img src="image_6.png" /><figcaption>**Figure 4.** pH-dependence of positive photoswitching. (A) mEos4b molecules immobilized in PAA gel (pH 6.4, 7.4 and 8) were photoconverted using 10 W/cm² 405 nm light and subsequently illuminated with 561 nm light (10 W/cm²). The fluorescence signal was recorded every 50 ms by illumination with 10 W/cm² 561 nm light for 5 ms. Shown is the mean ± sd of 4 measurements. (B) Photoconverted mEos4b molecules were diluted in universal buffer (pH 5 to 9) to an OD ~0.3. Samples were illuminated with ~40 mW/cm² 488 nm light to promote R1 to R2 switching for 12 minutes after which the molecules were allowed to thermally recover. Absorbance was integrated over the range 550-600 nm. The fluorescence was monitored every second by exposure to ~2 mW/cm² 561 nm light for 100 ms. Fluorescence emission was integrated over the range 570-850 nm.</figcaption></figure>

## R1 and R2 display different pKa's

A plausible scenario consistent with the results presented above is that R1 and R2 would display similar chromophore conformations, but would differ in the protonation state or H-bonding network of the chromophore environment. As a consequence, both R1 and R2 would exhibit similar fluorescent anionic states and nonfluorescent neutral states, but with different populations of anionic and neutral species (different pKa's). Because R1 builds up to homogeneity at every pH in the dark, its pKa can be safely extracted from a pH series of fluorescence spectra

(Figure S12A). To estimate the pKa of R2, we measured the absorbance and fluorescence switching kinetics of mEos4b over a wide pH range (Figure 4B) and performed a global fit using the simplified model of Figure S12B (see Supplementary Note 1 for further details). A pKa of ~7.7 could be estimated for R2 (Figure S12C, Table S2). In addition, the fit provided estimates for the switching quantum yields between R1 and R2 in their neutral states and the fluorescence quantum yield of R2. The fluorescence quantum yield of R2 was estimated to be 0.58, which is ~20% lower than R1. Although these values should be considered with care in view of the various assumptions made for the model fitting (Supplementary Note 1), the experimental pH-dependent switching behavior of Figure 4A could be nicely recapitulated using the SMIS simulator⁴⁵ with the derived photophysical model, providing qualitative confidence in the results (Figure S13). Overall, the data suggest that R2 behaves as a weakly fluorescent state at physiological pH and becomes completely dark at pH < ~6.

## Conformational changes between R1 and R2

To pinpoint the location of conformational (or chemical) differences between R1 and R2, we compared the NMR peak intensities of R1 measured under thermal equilibrium conditions (pure R1) and in a 405-nm illuminated sample (R1-R2 mixture). The intensity ratio serves as an indirect measure of NMR chemical shift and line width changes between the R1 and R2 states that could not be measured accurately from the recorded 2D spectra (Figure S6A) due to the limited R2 population (~35%) and highly overlapping R1 and R2 peaks for many residues. For amide sites unaffected by the conformational change (overlapping peaks, similar line widths), we expect an intensity close to 1. Conversely, for amide sites experiencing distinct chemical environments (NMR chemical shifts), the intensity ratio should decrease to the remaining R1 population, i.e. ~0.65 (Figures 5A and S14). Our assumption is comforted by the observation that residues with intensity ratios > 0.8 are all located in loop regions outside the β-barrel structure, where no changes in conformational dynamics (and thus chemical shifts) are expected. Highlighting the residues with the smallest intensity ratios (largest chemical shift changes) on the crystal structure of red mEos4b revealed that the difference in conformation is most significant in the α-helix preceding the chromophore and the four β-strands β2, β3, β10 and β11 facing His62 of the chromophore (Figure 5B). Interestingly, the involved region is essentially the same as that affected by the A/B exchange in the green-form, facing His62 of the chromophore and comprising the important tetrad Glu144-His194-Glu212-Gln38 which changes its hydrogen bonding network between the A and B states (Figure 5C).¹⁸ In the R1 state His62 is protonated at the ND1 position (similar to the green B state), while the hydrogen-bonding network formed by the Glu144-His194-Glu212 motif appears highly dynamic as indicated by extensive NMR line broadening observed for amide protons of residues 142-144, 194-196, and 211-214 (data not shown). Unfortunately, due to low NMR signal from these sites in the R2 (and R1) state, we could not determine whether His62 and Glu212 undergo a protonation change when switching between R1 and R2. Still, based on the observation that the same β-barrel region is involved in the conformational changes, and in view of the low intensity ratio (~0.6) observed for Glu212 (Figure S14) we speculate that similar proton rewiring may occur upon interconversion between the R1 and R2 states as occurs in the green form when switching between the A and B states.
<figure><img src="image_7.png" /><figcaption>**Figure 5.** NMR characterization of conformational changes in R2. NMR peak intensity changes observed during thermal relaxation from the 405 nm induced mixed (R1 and R2) to the thermally relaxed R1 state. (A) Residue specific intensity ratios are plotted as black bars. In addition, open green bars present the results of a global fit of residue specific kinetic R2-to-R1 thermal relaxation data (Figure S14) to a mono-exponential buildup function. (B) Residues with highest or lowest intensity ratios are color-coded on the crystal structure of red mEos4b (6GPO): red (> 0.8) and blue (≤ 0.65), while unassigned (undetected) residues in this region are color-coded in black. (C) Zoom on the chromophore and the Q38-E212-H194-E144 tetrad that plays an important role in mEos4b conformational heterogeneity.</figcaption></figure>

### Positive photoswitching in other green-to-red PCFPs

We investigated whether positive switching is a common property of green-to-red PCFPs. To this end, we monitored the behavior of various PCFPs by fluorescence imaging in PAA samples (Figure S15). Positive switching could only be seen in mEos-like PCFPs, such as pcSTAR and mEosEM, but not in other PCFP variants such as Dendra2,46 mEos4b-V69T (a Dendra2-like mutant)36, mkikGR,47 V127T SAAsoti 48 or LEA.49 These results suggest that, in those latter variants, either R2 does not form, or its formation is obscured by fast negative photoswitching. This is for example likely the case for LEA, for which positive switching was reported29 and could be verified spectroscopically (Figure S16A-B). In contrast, for mEos4b-V69T, the absence of positive switching could be confirmed with spectrophotometry (Figure S16C-D). It is interesting to note that in variants displaying primed photoconversion.37,50 here Dendra2 and mEos4b-V69T, E212 establishes a salt bridge with R66, stabilizing its protonation state.36 This observation is consistent with the notion that Glu212 may play a role in mEos4b positive switching.

### Impact of the R2 state on SMLM imaging

During PALM, sptPALM or Minflux data collection, a mEos4b molecule, once photoconverted to the red-form, may stochastically switch between R1 and R2 when illuminated with violet, cyan or green/yellow light. Depending on the experimental pH, this could produce intensity changes or apparent off times along the fluorescence trace. To assess whether R2 formation could interfere with PALM imaging, we acquired single molecule data of mEos4b at pH 6.5 and 7.5 and compared its blinking

behavior. The data show significantly more short-lived blinks at low pH (Figure S17), which we ascribe to R2 formation. Using SMIS simulations, we checked that the observed increase in blinking does not result from an increase in missed localizations due to the decreased mEos4b brightness at low pH (Figure S18). Thus, we conclude that R2 formation likely contributes to mEos4b blinking at the single molecule level, at least at low pH.

### DISCUSSION

This work reveals a novel photoswitching mechanism in photoconvertible fluorescent proteins of the EosFP family. Our investigations by NMR, spectrophotometry and fluorescence imaging lead us to conclude that this mechanism likely results from the interconversion between two red fluorescent states, R1 and R2, with different pKa's and brightnesses, resulting in the positive switching behavior recently noticed by others.28,29 As a consequence, R2 behaves as an apparent dark state at or below physiological pH. While only R1 is populated at thermal equilibrium, R2 builds up through light-induced proton rewiring of the chromophore environment. We propose that R1 and R2 are structurally and spectroscopically similar but display different pKa's because of electrostatic coupling between the chromophore and the surrounding protein matrix, a common feature in fluorescent proteins.51

An updated photophysical scheme of mEos4b is presented in Figure 6A and a simplified energy diagram focusing only on the R1-R2 switching mechanism is presented in Figure 6B. Proposed chromophore structures involved in negative ($R_{trans}$) and positive (R2) photoswitching are shown in Figure 6C. Starting from R1, excitation of the neutral chromophore with 405 nm or 488 nm light promotes formation of R2. Due to the high pKa of R2, this results in an increased fraction of neutral chromophores and a decreased fraction of anionic chromophores at physiological pH, lowering the fluorescence signal. Upon absorption of 561 nm light by the anionic chromophore of R2, back switching to R1 occurs and the fraction of anionic chromophores increases again while that of the neutral chromophores decreases. Concomitantly, 561 nm light induces dark state formation through *cis-trans* isomerization of the chromophore or excited state electron transfer giving rise to short-lived radicals. Those dark states are depopulated by thermal relaxation and by 405 or 488 nm light as documented earlier.15,21 Because the fraction of anionic chromophores in the R2 state decreases with pH, the effective light-induced back switching rate to R1 also decreases with pH, increasing the lifetime of R2 and its detrimental effects on the overall fluorescence. Importantly, since only partial R2 formation is observed under steady state illumination at or above physiological pH, excitation of the R2 neutral state with 405 or 488 nm light must also elicit switching to R1, albeit with lower efficiency. Likewise, we posit that the anionic R1 state is able to switch to R2 under 561 nm light with low efficiency. Of note, due to the CW lasers and power densities used in this work (typically ≤ 10 W/cm²), we exclude the possibility for any two-photon effect in these processes.

Proton rewiring upon chromophore excitation has been described for a variety of fluorescent proteins, although it is typically transient.52–54 In particular, proton transfer was described based on multi-site models in which the chromophore is energetically coupled to secondary protonation sites.23,55,56 Here, the exact excited-state reaction pathways leading to thermally metastable proton rewiring in red mEos4b remains to be established. The simple picture however is that, because excitation of the chromophore to the S1 electronic state shifts electron
density from the hydroxybenzylidene moiety to the imidazolium moiety, substantial electrostatic changes occur that may favor crossing free energy barriers between protonation states that cannot easily exchange in the dark. While in the green-form a change in the H-bond network linking the essential tetrad of amino acids Glu144-His194-Glu212-Gln38, together with a change in the protonation state of His62, was demonstrated to be at the core of proton rewiring, in the red form it remains unknown which protonation changes exactly take place. However,

we speculate that a change in the protonation state of the essential Glu212 might be involved. This would be also in line with the highly dynamic nature of this residue as noticed in several crystallographic studies.<sup>18,36,46,57</sup>

<figure><img src="image_8.png" /><figcaption>**Figure 6.** Updated photophysical model of EosFP-like PCFPs including R1-R2 transitions. (A) New simplified scheme based on this work, to be compared to **Figure 1**. The green-form of mEos4b exists in two conformations (A and B) at thermal equilibrium (for details, see **Figure S1**). The red-form, on the other hand, exists in a single conformation at thermal equilibrium (R1). However, under illumination, a second form (R2), with an increased pKa, appears. Dark refer to non-fluorescent *trans* and radical states. Dotted lines refer to thermal relaxation. (B) Energy diagrams representing R1 to R2 (left), and R2 to R1 (right) switching at physiological pH (pH 7.5). At this pH, R1 is mostly anionic, while R2 is slightly more protonated. In the ground state of R1, the energy barrier is too high to thermally switch to R2. In the excited state of R1, the energy barrier is reduced and R2 can be reached from both the neutral and anionic states. In the ground state of R2, the energy barrier to go back to R1 is low enough to be crossed with only thermal energy. Hence, R1 is energetically favored and fully populated at thermal equilibrium. In the excited state of R2, the energy barrier is further reduced. (C) Chromophore conformations. At thermal equilibrium the chromophore adopts a *cis* conformation (R1), which is mostly anionic at physiological pH. The R2 state maintains a *cis* conformation but is partly protonated at physiological pH. The R<sub>trans</sub> states (accessible from R1 or R2) have a *trans* conformation fully protonated at physiological pH. The thicknesses of the arrows reflect the relative rates and the colored backgrounds highlight the dominant protonation states at physiological pH.</figcaption></figure>

An important finding that emerges from our NMR data is that R2 itself is structurally heterogeneous, notably at low pH (Figure S10). Although the basis for this heterogeneity remains to be clarified, we propose that light-induced protonation changes in the chromophore environment of mEos4b may follow multiple pathways, similarly to the multiple proton wires previously reported in GFP.<sup>55</sup> The propensity to follow each pathway may vary as a function of pH, and, at low pH, different conformations might get stabilized or other conformations might become accessible. We do not exclude that minor species such as e.g. a *trans* cationic chromophore<sup>58</sup> may also form at low pH,

contributing to conformational heterogeneity of R2. Thus, rather than fixed energy barriers separating R1 and R2, multiple barriers might be involved.

Another interesting finding is that the brightness of R2 is lower than that of R1 (Table S2). Although there are no large structural rearrangements between R1 and R2, rewiring of the H-bond network around the chromophore induces electrostatic changes. This observation is consistent with the proposal that electrostatics in addition to steric constraints control fluorescence quantum yield in GFP variants.<sup>59</sup> Additionally, the dynamic and heterogeneous nature of R2 could also contribute to its reduced brightness.
Conformational heterogeneity in FPs,¹⁸,⁶⁰,⁶¹ resulting from small changes in the protein matrix while the chromophore structure stays essentially unmodified, may generate multiple fluorescent states in FPs. This emerging picture has now been described several times with various consequences on negative photoswitching.¹⁸,⁶²⁻⁶⁴ Positive switching in mEos4b is a novel manifestation of conformational heterogeneity.

Importantly, the equilibrium between states underlying confor-
mational heterogeneity may be different in the dark or under
light illumination, as different free-energy transition barriers are
crossed, depending on whether ground state or excited state
pathways are taken. Together with our previous investigation of
the mEos4b green form,¹⁸ this work provides an interesting ex-
ample: whereas the green form displays heterogeneity in the
dark, the red form only displays light-induced heterogeneity.
However, in both cases the structural ground for heterogeneity
is likely similar: proton rewiring in the chromophore environ-
ment. While dark or light-induced conformational heterogene-
ity may involve similar states, in practice, it is important to
make a distinction. For example, studying conformational dy-
namics of a given FP only at thermal equilibrium could be
highly misleading, as pronounced heterogeneity may only ap-
pear under light (and particularly those illumination conditions
that are employed in microscopy), as demonstrated in this work.
Furthermore, conformational heterogeneity can give rise to
complex scenarios, where the reached equilibrium may depend
in a subtle manner both on environmental (e.g. pH) and light-
illumination (e.g. balance between different wavelengths and
power densities) conditions. For example, in HEPES buffer R2
builds up to only ~35% at physiological pH under sole 405 nm
light while cis-trans isomerization under sole 561 nm light at
this pH depletes R1 by > 95%. At pH < 5.5 and in universal
buffer, however, R2 builds up to near 100% under cyan light.
The populations of the different dark states thus result from both
the physicochemical and illumination conditions.

Pinpointing the existence of multiple FP states in equilibrium
generally does not inform on phototransformation mechanisms.
A number of investigations on transformations such as photo-
conversion,⁶⁵ photoswitching,⁶⁶ radical state formation²⁴ or pho-
tobleaching³³ have revealed multiple intermediates occurring
on multiple time scales. Conformational heterogeneity adds up
to, and in fact may be intertwined with such complexity. Indeed,
different conformers may undergo phototransformations with
different mechanisms and/or at different rates.

Another important consequence of this work is that R2 for-
mation reduces the effective brightness of mEos4b and likely
contributes to its blinking behavior at the single-molecule level,
the more so at low pH. This adds a new mechanism at the origin
of short off-times in PCFPs, in addition to those assigned to rad-
ical formation.²⁰,²¹ Interestingly, because of the intense 561 nm
(~ 1 kW/cm²) and weak 405 nm (~ 1 W/cm²) power densities
typically used in PALM, R2 formation may principally occur
from the anionic R1 state under these conditions (**Supplemen-
tary Note 2**). This is in contrast with the situation for ensemble
measurements, as used in this work, where both lasers are used
at similar power densities and R2 mostly originates from the
neutral R1 state. Thus, we propose that even at high pH (pH >
~7) there is frequent R2 formation in PALM. However, the con-
sequences are expectedly small because at such pH R2 is largely
anionic, hence fluorescent, and very short-lived. In contrast, at
lower pH (pH < ~7), R2 is mostly protonated, hence nonfluo-
rescent, and longer lived. Consequently its formation results in

the appearance of detectable blinks and reduced photon budget
per localization.

Another consequence of our study concerns the estimation of extinction coefficients of the red-form in EosFP variants. Estimating these extinction coefficients is notoriously difficult due to the added effect of photobleaching during photoconversion and partial overlap of the absorption bands of the anionic green state and neutral red state.³ The presence of R2 adds another layer of complexity to the measurements. As clearly seen in **Figure 2C**, the sole application of 405 nm light to promote photoconversion, typically near pH 7, results in a significant fraction of R2 to accumulate in the protonated form. This fraction does not contribute to the measured absorbance of the anionic chromophore near 580 nm, resulting in significant underestimation of the associated extinction coefficient. Therefore, extinction coefficients reported in the literature and FPbase⁶⁷ may need to be revisited taking advantage of thermal relaxation in the dark or of 561 nm light to fully populate R1. We note that, for similar reasons, attempts to measure photoconversion efficiency (PCE) in EosFP variants using sole 405 nm light would lead to strongly underestimated values (**Supplementary Note 3**).

CONCLUSION

While there has recently been considerable progress in the de-
sign of performant organic dyes for super-resolution micros-
copy approaches, the expanding field of live cell nanoscopy
suggests that green-to-red PCFPs will remain fluorophores of
choice in modalities such as sptPALM or Minflux. Although
these techniques require exquisite comprehension of the com-
plex photophysical behavior of these markers, available
knowledge remains limited. In this work, we focused on the
photophysics of mEos4b in its red-form. We demonstrated that,
in contrast to the green-form, the red-form of this PCFP and
related variants is homogenous at thermal equilibrium. How-
ever, heterogeneity is restored by light, promoting the formation
of a second red-form, characterized by a high pKa and a lower
brightness, in a pH-dependent manner. The resulting positive
photoswitching behavior observed here with mEos4b is likely
the same as that recently observed for mEos3.2²⁸ and LEA.²⁹
The underlying mechanism is proposed to involve light-induced
protonation changes within the fluorescent protein matrix flank-
ing the His62 imidazole chromophore moiety. Positive pho-
toswitching in EosFP variants is suggested to induce hitherto
undescribed short-lived dark states in single-molecule localiza-
tion experiments. This work highlights the complex relationship
between the photophysical behavior and free-energy landscapes
of fluorescent proteins both in the ground and excited state.

ASSOCIATED CONTENT

**Supporting Information.** Protein production, Sample preparation, NMR, EPR spectrophotometry, single-molecule imaging, global fitting and photophysical simulations (Materials and Methods); global fitting, estimation of photoconversion efficiency, R2 formation (Notes S1–S3); additional photophysical schemes, NMR, spectroscopic, and ensemble and single-molecule fluorescence data, simulated data and setup description (Figures S1–S18); and
NMR chemical shifts, fitted parameters, photoconversion efficiencies and parameters used in simulations (Tables S1–S4). This material is available free of charge at https://pubs.acs.org/doi/xxxx.

# AUTHOR INFORMATION

## Corresponding Author

Dominique Bourgeois – Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 38000 Grenoble, France ;
https://orcid.org/0000-0002-1862-7712;
Email: dominique.bourgeois@ibs.fr

Bernhard Brutscher – Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 38000 Grenoble, France ;
https://orcid.org/0000-0001-7652-7384;
Email: bernhard.brutscher@ibs.fr

## Authors

Jip Wulffelé – Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 38000 Grenoble, France ; https://orcid.org/0009-0009-6337-7378

Arijit Maity – Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 38000 Grenoble, France ; https://orcid.org/0000-0002-2399-6600

Isabel Ayala – Univ. Grenoble Alpes, CEA, CNRS, Institut de Biologie Structurale (IBS), 38000 Grenoble, France

Serge Gambarelli – Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, Grenoble F-38000, France, https://orcid.org/0000-0002-7204-7022

## Author contributions

Jip Wulffelé and Arijit Maity contributed equally to this work.

## Funding Sources

Financial support from iNEXT-Discovery (871037) funded by the Horizon 2020 program of the European Commission, the Agence Nationale de la Recherche (grants no. ANR-20-CE11-0013-01, ANR-22-CE13-0046 and ANR-22-CE11-0011-01, and the IR INFRANALYTICS (FR2054) is acknowledged. This work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).

## Notes

The authors declare no competing financial interest.

# ACKNOWLEDGMENT

The authors acknowledge Virgile Adam and Oleksandr Glushonkov for support with the design of the in situ NMR spectrophotometer, the setup of our home built microspectrophotometer and the IBS cell imaging M4D platform, Adrien Favier and Alicia Vallet for support on the IBS NMR platform, Matthieu Sainlos and Loris Verron for providing plasmids for mEos4b surface immobilization

with the SpyCatch/SpyTag technology and Salvatore de Bonis and Daniel Janzen for help with protein production and purification.

# ABBREVIATIONS

PCFP: photoconvertible fluorescent protein; RSFP: reversibly switchable fluorescent protein; SMLM: single molecule localization microscopy; EPR: electron paramagnetic resonance
NMR: nuclear magnetic resonance; PALM: photoactivatable localization microscopy; sptPALM: single particle tracking PALM; MinFlux: Minimal Fluorescence Photon Fluxes Microscopy

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