Photophysics of the red-form Kaede chromophore

Abstract

The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.

Fig. 1. Photoconversion of the chromophore in Kaede. The principal GFP chromophore (a, HBDI) is present in the green form of Kaede, which photoconverts under UV irradiation to the red form (b, rK1H).

Fig. 2. Absorption spectra for: (a) rK1H, and (b) rK1⁻ as a function of solvent: MeCN; butan-1-ol (BuOH); ethylene glycol (EtG); ethanol (EtOH); THF.

Fig. 3. Solvent-dependent emission spectra: (a) rK1H, and (b) rK1⁻. Spectra are normalised for constant absorbance to reveal the relative quantum yield of fluorescence. Excitation was at 420 nm.

Fig. 4. Relative fluorescence intensity from rk1H (open symbols) and rK1⁻ (solid symbols) as a function of the viscosity of ethanol:ethylene glycol mixtures.

Fig. 5. Ultrafast time-resolved fluorescence of rK1H excited at ≈400 nm: (a) fluorescence decay profiles recorded in a range of solvents (EG = ethylene glycol; F = formamide). Note that data are presented on a logarithmic intensity scale to highlight the non-single exponential decay. (b) Emission wavelength dependence of the decay profile in methanol. The insets in (b) show the early time data and the wavelengths spanned by the measurement (470 nm–580 nm) respectively. Fitting parameters from the analysis of these data are given in the ESI.

Fig. 6. Transient absorption spectroscopy for rK1H and rK1⁻. (a) Heat map for rK1H in ethanol excited at 440 nm. Blue indicates ground state bleach plus stimulated emission, and red/yellow indicates the excited-state absorption. (b) DADS from a global analysis of the data in (a) using the sum of two decay components. (c) DADS for rK1⁻ (see heat map in the ESI, Fig. S6). In (a), (b), and (c) the scattered light contribution is shaded out. (d) The recovery kinetics at specific wavelengths, reflecting the excited state decay and ground state recovery in ethanol and ethylene glycol (solid lines are the global fits).

Fig. 7. FSRS spectra of (a) rK1H, and (b) rK1⁻ in methanol. Spectra were measured as a function of time after an actinic pulse (440 nm for rK1H and 535 nm for rK1⁻) with Raman pulses at different wavelengths (700 nm for rK1H and 650 nm for rK1⁻). The vertical stick spectra (orange) at the bottom of panels (a) and (b) show the TDDFT calculated excited state Raman spectra. (c) The DFT calculated ground state Raman spectra for the two forms are shown for reference.

Fig. 8. Temperature dependence of the fluorescence emission spectra for (a) rK1H and (b) rK1⁻ in ethanol.

Fig. 9. Franck–Condon–Herzberg–Teller simulations of the absorption (black) and emission (red) spectra, at T = 0 K, for (a) rK1H and (b) rK1⁻. The 0–0 transition has been shifted to match the experimental spectra.

Fig. 10. Schematic potential energy surfaces (blue lines) illustrating the origins of observed complex photophysics of rK1H and rK1⁻. A single ground state conformer exists, but with a distribution of geometries (including solvent molecules) around the average conformation due to a shallow potential energy surface. This distribution is broader at higher temperature (grey Gaussian lines). Electronic excitation transfers this distribution to the Franck–Condon excited state. The excited state surface is more complex, with multiple minima separated by low barriers. The broad distribution relaxes in <100 fs to directly populate different minima, where each decays with its distinct fluorescence spectrum (down arrows) and has a lifetime determined by its pathway and energy barrier to a conical intersection (CI) at which internal conversion occurs. At low temperature, the ground state distribution is narrowed and only a single excited state minimum is populated from the Franck–Condon state, which then decays with a long but single exponential decay time.