Decoupling Electronic versus Nuclear Photoresponse of Isolated Green Fluorescent Protein Chromophores Using Short Laser Pulses

The photophysics of a deprotonated model chromophore for the green fluorescent protein is studied by femtosecond laser pulses in an electrostatic ion-storage ring. The laser-pulse duration is much shorter than the time for internal conversion, and, hence, contributions from sequential multiphoton absorption, typically encountered with ns-laser pulses, are avoided. Following single-photon excitation, the action-absorption maximum is shown to be shifted within the $S_0$ to $S_1$ band from its origin at about 490 to 450 nm, which is explained by the different photophysics involved in the detected action.

Figure 1

The structure of the deprotonated $p$-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) anion used as a GFP model chromophore in the present work. Two resonance forms of the anion are shown.

Figure 2

Schematic of the experimental setup comprising the electrospray-ion source with the cooled ion trap, the SAPHIRA ion-storage ring with particle detectors DET 1 and DET 2, and the fs- and ns-laser systems.

Figure 3

Counts of neutral fragments on DET 1 in SAPHIRA as a function of time, where time zero is the laser-firing time. Top: Data obtained with the ns laser (480 nm, 1.2 mJ). Excess counts of neutrals are detected after the laser is fired. In a time window termed “Prompt,” early events from the first revolution are registered, and later events are detected in a time window termed “Delayed.” Analysis based on Poisson statistics for sequential independent absorption events verifies that the delayed action arises from two-photon absorption solely. The action in the prompt window is at low pulse energy primarily due to single-photon absorption (for details, see the Supplemental Material [18]). Bottom: Data obtained with the fs laser (480 nm, 0.1 mJ), where only action in the prompt window is observed. The ion trap is at room temperature (300 K).

Figure 4

Schematic presentation of the molecular response after photoabsorption with the electronic response, prompt electron autodetachment, and nuclear response through internal conversion back to a hot electronic ground state (IC). The sequential absorption of multiple photons requires a laser pulse duration longer than the ps-time scale for IC. Dissociation out of the ground state after absorption of a single photon ($n=1$) is very slow and, hence, not detectable, whereas it becomes detectable after two-photon absorption (Fig. 3).

Figure 5

Action absorption spectra obtained with ns- and fs-laser pulses. Red data were recorded with the ion trap at room temperature (300 K) and blue data were recorded at 100 K. Panel (a) Action cross section registered in the prompt time window from DET 1 with the ns laser. At 100 K, data were obtained with high (bold data points) and low (open data points) laser-pulse energy. Panel (b) Action cross section registered in the delayed time window from DET 1 recorded with the ns laser. Panel (c) Prompt action from DET 1 recorded with the fs laser. Panel (d) The theoretical absorption cross section where the electronic continuum and the 0–0 transition of the $S_1$ band are indicated. Red curve 300 K; blue curve 100 K. The computational details are described in Ref. [21].