# Cryogenic action spectroscopy of the cyan fluorescent protein chromophore anion

Jordan Dezalay, Eleanor K Ashworth, Jack E Fulker, Mark H Stockett,

Jennifer A. Noble, James N Bull

► To cite this version:

Jordan Dezalay, Eleanor K Ashworth, Jack E Fulker, Mark H Stockett, Jennifer A. Noble, et al. Cryogenic action spectroscopy of the cyan fluorescent protein chromophore anion. Physical Chemistry Chemical Physics, 2025, 27 (32), pp.16738 - 16743. 10.1039/d5cp02520f. hal-05382362

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Cite this: Phys. Chem. Chem. Phys.,
2025, 27, 16738

Received 2nd July 2025,
Accepted 16th July 2025

DOI: 10.1039/d5cp02520f

rsc.li/pccp

Action spectroscopy at *T* ≈ 30 K, as a proxy for the visible absorption band, and the branching between electron detachment and dissociation in the cyan fluorescent protein chromophore anion are reported. The cryogenic action spectra, which show the presence of several rotamers, serve as a reference point for interpreting the effect of nano-environmental interactions in complex protein environments. The adiabatic detachment energy for the lowest energy geometric isomer (Z1) is 19 531 ± 40 cm⁻¹, with the vertical S₁ ← S₀ transition energy at 23 734 ± 40 cm⁻¹. For Z1, the propensity for internal conversion followed by dissociation is low (<10%) compared with autodetachment as the S₁ ← S₀ absorption band is entirely situated in the detachment continuum and is classified as a shape resonance.

Fluorescent proteins (FPs) are the foundation stone for modern, high-resolution bioimaging of cells and the monitoring of microscopic biological processes.¹ Accordingly, a wide range of derivatives of FPs have been developed offering a full colour palette,²⁻⁵ including cyan fluorescent protein (CFP).⁶ For CFP, mutations over three generations of FPs have substantially improved quantum yield and other fluorescence properties,⁷⁻⁹ allowing it to become a widespread biomarker. In several derivative CFPs, including NowGFP and mNeonGreen,¹⁰,¹¹ the chromophore may be deprotonated and probably resides as the anion (cyan⁻). The optical properties of FPs are dictated by the chromophore unit embedded within a protein binding pocket, with the amalgamation of non-covalent interactions and the local electrostatic field leading to an electrochromic spectral shift and perturbation of the intrinsic photophysics of chromophores.¹²,¹³ It is of fundamental importance to characterise the photophysics of FP chromophores in a ‘bottom up’ approach in order to understand

the influence of the environment (solvent or protein)¹⁴ and for calibrating or benchmarking of theory.¹⁵

The chromophore in CFP, shown in Fig. 1, is a tryptophan-based derivative of the well-known green fluorescent protein (GFP) chromophore. By probing FP chromophores in isolation (i.e. *in vacuo*), their intrinsic photophysics can be elucidated without the complications of solvent effects (e.g. solvatochromic shifts).¹⁶ While the photophysics of the anionic GFP chromophore and substituted forms has been studied thoroughly in the gas phase using action spectroscopy techniques at *T* ≈ 300 K,¹⁷⁻²⁵ and at cryogenic temperatures,²⁶⁻²⁹ and is understood in the gas phase through *ab initio* molecular dynamics simulations,¹⁵,³⁰ the anionic CFP chromophore is

<figure><img src="image_3.png" /><figcaption>**Fig. 1** (a) Structure of the cyan chromophore (red) within CFP, which adopts a Z2 rotamer (i.e. rotation about the central single bond) configuration due to an array of non-covalent interactions. (b) Lowest energy rotamer of isolated cyan⁻ (blue), denoted as Z1, which has Z-isomer geometry about the methylene bond. For simplicity, we refer to all geometric isomers and rotamers (Z1, Z2, E1, and E2) as isomers. Structures of all isomers are summarised in the ESI.†</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<footnote>ᵃAix-Marseille University, CNRS, PIIM, Marseille, France</footnote>

<footnote>ᵇChemistry, Faculty of Science, University of East Anglia, Norwich NR4 7TJ, UK.
E-mail: james.bull@uea.ac.uk</footnote>

<footnote>ᶜDepartment of Physics, Stockholm University, SE-10691 Stockholm, Sweden</footnote>

<footnote>†Electronic supplementary information (ESI) available: Mass spectra demonstrating anion fragments, optimised geometries of possible cyan⁻ isomers and rotamers. See DOI: https://doi.org/10.1039/d5cp02520f</footnote>
largely unstudied apart from ion storage ring measurements³¹,³² and room-temperature anion photoelectron spectroscopy.³³ It is valuable to perform clear gas-phase experiments on mutant GFP chromophores in order to understand structure-activity trends in the photoactive units of fluorescent proteins.

Here, we characterise the S₁ ← S₀ absorption band and adiabatic electron detachment threshold of isolated cyan⁻ at T ≈ 30 K using action spectroscopy, monitoring photoneutrals (parent and fragment neutrals). Our measurements used the cryogenic ion trap action spectroscopy apparatus at the PIIM laboratory (Aix-Marseille University),³⁴,³⁵ where cyan⁻ was generated through electrospray ionisation (at T = 300 K) of a 1:1 methanol-water solution with a trace amount of ammonia. Significantly, because the chromophore was synthesised stereospecifically as a Z-isomer,²⁵ it was assumed to be predominately

the Z isomer in the gas phase based on the use of 'gentle' ion production conditions.²¹ Electrosprayed ions were trapped in a 3D quadrupole ion trap cooled using a helium cryostat; several temperature sensors spread across the ion trap assembly measured temperatures ranging from 12 to 35 K,³⁶ with the contents of the trap expected at T ≈ 30 K (the minimum temperature measured close to the cold head was T = 21 K). Since the action spectroscopy is dominated by electron detachment, with dissociation being a minor channel, we performed action spectroscopy monitoring parent and fragment neutrals. Here, trapped parent anions were extracted and irradiated (3.62 µs after extraction) with the laser radiation (EKSPLA NT-342B optical parametric oscillator, ≈6 cm⁻¹ spectral resolution, calibrated with a wave-meter) in a Gauss tube as part of a time-of-flight region. Parent and fragment anions were decelerated, allowing neutral particles

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Action spectra of cyan⁻ recorded at T ≈ 30 K: (a) complete spectra recorded for the parent neutral signal (black) and fragment neutrals (red), with a step size of 0.5 nm. (b) Higher resolution scan (black) over the main group of transitions, with a step size of 0.2 nm. Franck-Condon-Herzberg-Teller (S₁ ← S₀) simulations for the Z1 (blue) and Z2 (orange) isomers are shown and were translated so that respective 0-0 transitions match the onset of the two spectral bands. (c) Higher resolution scan (0.06 nm step size) over region A, assigned to the weak signal from E1 (green) and E2 (magenta) isomers. The inset corresponds to fragment neutrals, with a threshold extrapolated to ≈20 900 cm⁻¹. * Denotes the simulated hot-band signal from E1, which correlates with the fragment neutral threshold (see the inset). (d) Extrapolation of the parent neutral signal to the adiabatic detachment energy (ADE) threshold at 19 531 ± 40 cm⁻¹. No fragment neutral signal was detected for *hv* < 20 900 cm⁻¹ (see the inset in c). (e) Calculated DBS orbital for the Z1 isomer with an isosurface value of 0.005 a.u. and a binding energy of 41 meV (≈ 330 cm⁻¹).</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /></figure>

to impact a microchannel plate detector. Because dissociation is accompanied by kinetic energy release (i.e. fragments moving away from each other), there is a change in the recorded time-of-flight (TOF) profile, allowing the fragment yield to be quantified by analysing laser-on against laser-off TOF profiles. Data were normalised relative to the laser-off signal and light fluence. A complete description of the experimental strategies is given in ref. 37.

To support assignments of the experimental spectra, quantum chemical calculations were performed. Geometries and vibrational frequencies were computed at the ωB97X-D/aug-cc-pVTZ level of theory³⁸,³⁹ using Gaussian 16.B01,⁴⁰ while electron detachment energies and vertical excitation energies (VEEs) were computed at the DLPNO-CCSD(T)/aug-cc-pVTZ⁴¹ and STEOM-DLPNO-CCSD/aug-cc-pVTZ⁴² levels of theory using ORCA 6.0.1.⁴³ Absorption profiles ($S_1 \leftarrow S_0$ transition) were simulated using the Franck-Condon-Herzberg-Teller (FCHT) framework as implemented in Gaussian.⁴⁴ The dipole-bound state (DBS) was characterised using the EOM-CCSD/aug-cc-pVDZ + 8spd methodology, where +8spd is a series of uncontracted functions (orbital exponents $\zeta = 0.1$ to $2.91 \times 10^{-5}$, with a geometric progression ratio of 3.13)⁴⁵ situated $\approx 5$ Å beyond the molecule along the dipole moment vector of the radical neutral (in the anion geometry).

Action spectra recorded over the 19 500–44 000 cm⁻¹ ($\approx 500–225$ nm) range are shown in Fig. 2a. Parent neutrals were produced in much higher abundance than fragment neutrals across the studied spectral range, demonstrating that autodetachment is much more probable than photoexcitation followed by internal conversion and statistical dissociation. The spectra reveal three bands, labelled A, B, and C, which are present in both parent neutral and fragment neutral spectra. These were assigned as follows: A – E1 and E2 isomers, B – Z2 isomer, and C – Z1 isomer. This assignment is consistent with the band intensities and expected gas-phase populations ($T = 300$ K Boltzmann distribution followed by rapid cooling and kinetic trapping). Specifically, from the relative energies in Table 1, a ratio of 100:73 for Z1:Z2 is predicted, which is in good agreement with the experimental band intensities. A small amount of E1 and E2 may be produced through collisions in low vacuum regions (the nascent population of E1 at $T = 300$ K is $\approx 3\%$).²¹ Additionally, the ordering of computed VEEs (Table 1) is consistent with the band assignments. For each of the four isomers, the $S_1 \leftarrow S_0$ transition is bright (oscillator strength $f = 0.6$ for Z1), while the VEE for the $S_2 \leftarrow S_0$ transition

<table><caption>**Table 1** Calculated energetics of each cyan⁻ isomer (see the illustrations in the ESI). Relative energy ($E_{rel}$), vertical excitation energy (VEE) for the $S_1 \leftarrow S_0$ transition, and adiabatic and vertical detachment energies (ADE and VDE) are all given in cm⁻¹. Dipole moment magnitudes, $|\mu|$, are given in Debye</caption><thead><tr><th>Species</th><th><i>E</i><sub>rel</sub></th><th>VEE</th><th>ADE</th><th>VDE</th><th>|<i>μ</i>|</th></tr></thead><tbody><tr><td>Z1</td><td>0</td><td>25 121</td><td>22 148</td><td>23 197</td><td>4.75</td></tr><tr><td>Z2</td><td>66</td><td>24 871</td><td>22 495</td><td>23 455</td><td>6.69</td></tr><tr><td>E1</td><td>720</td><td>24 759</td><td>22 301</td><td>23 197</td><td>6.10</td></tr><tr><td>E2</td><td>2013</td><td>24 994</td><td>22 648</td><td>23 479</td><td>6.49</td></tr></tbody></table>

is calculated in the range of 27 000–30 000 cm⁻¹ but with $f \approx 10^{-2}$.

A higher resolution spectrum of the parent neutral over the $S_1 \leftarrow S_0$ transition is shown in Fig. 2b. There is no obvious vibrational structure, which is in parallel with observations for action spectra of protonated cations of cyan similarly recorded at $T \approx 20$ K. FCHT simulations predict the transitions for the Z1 and Z2 isomers to be origin dominated, although the overlap of spectra due to multiple isomers complicates an experiment-theory comparison. Our FCHT simulations, included in Fig. 2b, indicate the spectrum should be dominated by contributions from a $\approx 70$ cm⁻¹ mode (torsion of the single bond on the methylene bridge). This, combined with higher frequency combination bands and indole ring modes, lead to a congested spectral profile. An earlier room-temperature photoneutral spectrum of cyan⁻ revealed a single peak with the wavelength of maximum response at $\approx 459$ nm ($\approx 21 790$ cm⁻¹),³¹ which is red shifted by $\approx 2000$ cm⁻¹ compared with the most intense band (C) present in the cryogenic spectrum (23 835 ± 50 cm⁻¹, Fig. 2). STEOM-DLPNO-CCSD calculations on excited states of the radical neutrals produced energies outside of our spectral window, consistent with no direct detachment channels that could account for bands A, B, or C. This result is consistent with the expected radical neutral excited states for the related green fluorescent protein chromophore anion.²⁰

The parent neutral spectrum recorded over band A is shown in Fig. 2c, revealing a clear vibrational structure starting at $\approx 21 100$ cm⁻¹. The spectral position of this vibrational structure was not consistent with any hot band simulations for the Z1 or Z2 isomers; the hot band signal extending from band C or B over band A would require ions with room temperature or warmer internal energies. However, FCHT simulations show that band A is consistent with small quantities of the E1 and E2 isomers (expected at $\approx 3\%$ from an initial Boltzmann thermal population). The FCHT simulations show that the E1 isomer is dominated by the origin and first excited vibration of the $\approx 62$ cm⁻¹ torsion mode, although the E2 isomer shows an extended progression dominated by a 62 cm⁻¹ mode, which is because of the steric hindrance and consequential non-planarity of the E2 isomer (leading to the highest $E_{rel}$ value in Table 1), i.e. the excited state for E2 is non-planar. The inset in Fig. 2c shows that the weak signal associated with fragment neutrals is observed from $\approx 20 900$ cm⁻¹, consistent with the assignment of band A to electronically excited anionic states rather than being linked with direct photodetachment processes. Assignments of the labelled vibrations in Fig. 2c are given in the ESI.†

While the fragment neutral signal threshold is $\approx 20 900$ cm⁻¹ (Fig. 2c, inset), the parent neutral threshold was extrapolated to a lower wavenumber of 19 531 ± 40 cm⁻¹ (Fig. 2d), which defines the adiabatic detachment energy (ADE). We assign this value to the Z1 isomer since it is the most abundant gas phase species and because the calculated ADEs for each isomer (Table 1) indicate that Z1 appears at the lowest energy. The calculated value (Table 1) is $\approx 2600$ cm⁻¹ higher in energy. Since the direct photodetachment signal is so weak compared with autodetachment from the overlapping $S_1 \leftarrow S_0$ transition, it is not possible to discern a reliable
Communication

VDE value. Room temperature photoelectron spectroscopy mea-
surements on cyan⁻ have shown VDE = 2.75 ± 0.02 eV (22 177 ±
161 cm⁻¹),³³ which is likely complicated by large autodetachment
signals considering the photon energies chosen in that study. It is
also worth noting that our fragment neutral signal threshold at
≈20 900 cm⁻¹ is consistent with the critical energy threshold
(kinetically shifted dissociation threshold) from a recent cryogenic
ion storage ring study.³²

TOF mass spectrometry confirmed that the neutral frag-
ments, which are produced in low quantity (<10%) compared
with the parent neutral signal, are linked with the production of
anions with m/z 223 (–CH₃) and m/z 153 (likely involves intra-
molecular rearrangement), with the former being the predomi-
nant photofragmentation pathway for the GFP chromophore
anions.²⁴,⁴⁶ The bond dissociation energy (BDE) for the –CH₃
channel is ≈1.6 eV (≈12 900 cm⁻¹),³¹ which is ≈6600 cm⁻¹
below the adiabatic detachment threshold. We consequently
expect that the small amount of S₁ population following
photoexcitation that is able to internally convert to recover
the ground electronics will undergo dissociation rather than
thermionic emission. In turn, this implies that the parent
neutral spectrum should be predominately from direct photo-
detachment and autodetachment processes.

It is worth noting that the parent neutral cores for each
isomer of cyan have permanent dipole moments that support a
(non-valence) DBS since |μ| > 2.5 D (Table 1).⁴⁷,⁴⁸ The calculated
DBS orbital for the Z1 isomer is shown in Fig. 2e with a calculated
binding energy of 41 meV (≈330 cm⁻¹). As expected, the orbital is
localised in the direction of the positive end of the molecular
dipole moment for the neutral core. Because the oscillator strength
to excite the DBS is *f* < 10⁻⁴, such states are only observed clearly,
e.g. the ground vibrational state through two-photon detachment⁴⁹
or collisional detachment,⁵⁰,⁵¹ when the spectroscopic focus is not
on nearby valence-localised states with oscillator strengths four
to five orders of magnitude higher (Z1, *f* ≈ 0.6 for the S₁ ← S₀
transition).⁵²,⁵³ Furthermore, cryogenic action spectroscopy on
deprotonated indole similarly showed no clear individual DBS
transitions in the vicinity of the detachment threshold (rather a
broad, unresolved distribution),⁵⁴ while other nitrogen-conditioning
PAHs show clearly resolved DBS vibrations. We conclude that DBS
transitions are very weak in cyan⁻ action spectra, are swamped by
the other excitation processes and are consequently not important in
the action spectroscopy of cyan⁻.

In some ways, the cryogenic action spectra of cyan⁻ contrast
with those recorded for the GFP chromophore anion. The latter
may exist as *E* and *Z* geometric isomers in the gas phase,²¹ but
is not complicated by rotamers due to the symmetry of a *para*-
phenoxide moiety rather than the deprotonated indole group in
cyan⁻. Moreover, the GFP case shows a clear vibrational struc-
ture with a pronounced ≈80 cm⁻¹ progression assigned to the
methine bridge bending/ring scissoring mode from FCHT
simulations.²⁷,²⁸ Significantly, in the case of the GFP chromo-
phore anion, the S₁ ← S₀ absorption band extends over the
detachment threshold with experiments showing differing
electron detachment to dissociation branching occurring when
tuning the laser wavenumber over the band due to a small

excited state barrier (≈250 cm⁻¹) to an internal conversion
coordinate.²⁶,²⁸ This interpretation has been further evidenced
by recent cryogenic gas-phase fluorescence measurements.²⁹ In
contrast, for cyan⁻, the electron detachment threshold (for Z1)
is situated in the range of 1500–2000 cm⁻¹ below the onset of
the S₂ ← S₁ transition. Thus, the S₁ excited state in cyan, which is
classified as a shape resonance, is entirely situated above the
detachment threshold. In such a case, we expect the shape
resonance lifetimes to be on the order of tens to hundreds of
femtoseconds and, consequently, to show lifetime broadening.¹⁶,⁵⁵
On the other hand, the weak electron detachment signal asso-
ciated with the *E*1 and *E*2 isomers (which is red-shifted compared
with the *Z*-isomers and the *E*-isomers have higher calculated
ADE values, Table 1) shows progression with simulated 62 cm⁻¹
spacing, consistent with twisting of the methine bridge and the
resolved vibrational structure due to the longer excited-state
lifetime.

In summary, cryogenic action spectroscopy of cyan⁻ has char-
acterised the S₁ ← S₀ absorption properties of the Z1, Z2, E1 and
E2 isomers and the adiabatic electron detachment thresholds for
the Z1 isomer. The lack of a clear vibrational structure, for the
major isomer, Z1, is attributed to some combination of congested
vibronic transitions and lifetime broadening effects due to prompt
autodetachment. The presence of two rotamers for the gas-phase
anion is expected to translate to solution, although the abundance
of each rotamer will depend on specific solvent-molecule interac-
tions. Consequently, experimental studies of the chromophore in
solution (e.g. using ultrafast pump-probe spectroscopies), will
probe differing conformer distributions depending on if the pump
excites on the red or blue edge of the absorption band.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data that support the findings of this study are available
from the corresponding author upon reasonable request.

Acknowledgements

This work was funded by an EPSRC New Investigator Award (EP/
W018691 to J. N. B.), a project grant from the Olle Engkvist
Foundation (200-0575 to M. H. S.), and the Swedish Foundation
for International Cooperation in Research and Higher Education
(STINT) Grant for Internationalisation programme (PT2017-7328
to M. H. S. and J. N. B.). E. K. A. thanks the University of East
Anglia for a doctoral studentship.

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