Power Density Titration of Reversible Photoisomerization of a Fluorescent Protein Chromophore in the Presence of Thermally Driven Barrier Crossing Shown by Quantitative Millisecond Serial Synchrotron X-ray Crystallography

Abstract

We present millisecond quantitative serial X-ray crystallography at 1.7 Å resolution demonstrating precise optical control of reversible population transfer from Trans-Cis and Cis-Trans photoisomerization of a reversibly switchable fluorescent protein, rsKiiro. Quantitative results from the analysis of electron density differences, extrapolated structure factors, and occupancy refinements are shown to correspond to optical measurements of photoinduced population transfer and have sensitivity to a few percent in concentration differences. Millisecond time-resolved concentration differences are precisely and reversibly controlled through intense continuous wave laser illuminations at 405 and 473 nm for the Trans-to-Cis and Cis-to-Trans reactions, respectively, while the X-ray crystallographic measurement and laser illumination of the metastable Trans chromophore conformation causes partial thermally driven reconversion across a 91.5 kJ/mol thermal barrier from which a temperature jump between 112 and 128 K is extracted.

Figure 1

SSX fixed target data collection optical setup, showing the illumination pattern (Top) of the 473 nm preillumination which accumulates the Trans state (black arrow on light blue), the 405 nm pump (dark blue) which generates the Cis state, and X-ray beam (red) as well as the free space focusing geometry (bottom).

Figure 2

Power density titration of Trans-to-Cis photoisomerization using 2 ms flashes at 405 nm, following 473 nm continuous wave accumulation of the Trans state. Q-weighted difference electron density relative to the 14.43 mJ/mm² illumination condition with dominating Cis state is plotted at ±3σ rms (positive in blue, negative is magenta), scaled by resolution bins. Cis coordinates are shown in cyan and Trans in yellow. Panels (a)–(e) show differences from the highest energy density (E = 14.43 mJ/mm²) structure factors: F_obs(E = 14.43) – F_obs(E). For comparison, panel (f) shows the difference of two steady state structures collected using MX reported previously.

Figure 3

Difference electron density for 0.53 mJ/mm² energy density: mF_obs (0.53) – nF_calcd (Occupancy). Plotted at ±2.0 rms with negative and positive features in magenta and blue, respectively. The percentage indicates the cis occupancy fraction. In the bottom right, the integrated negative electron density around the chromophore is plotted as a function of the Cis occupancy with the minimum indicated.

Figure 4

Left: Population fraction of the Cis state is plotted as a function of energy density state for the different occupancy determination methods. The fluence-based first-order rate constants, k(mm²/mJ) and retrieved temperature jump values are reported for the four different crystallographic quantification methods. Right: (a) (0 mJ/mm²), (b) (0.10 mJ/mm²), (c) (0.53 mJ/mm²), (d) (1.78 mJ/mm²), (e) (6.74 mJ/mm²), (f) (14.43 mJ/mm²) phased electron density maps at 1.5 rms using occupancies determined by minimization of 2F_obs – F_calcd density with increasing energy density of the 405 nm 2 ms flashes. A clear shift in electron density can be seen toward the photoproduct (Cis state) as the energy density increases. The crystallographic determinations of Cis population at zero energy density were 0.56 ± 0.05 (R_Free method), 0.33 ± 0.05 (F_extrapolated), 0.25 ± 0.05 (R_Work), and 0.29 ± 0.05 (F_o – F_c).