Observation of Cation Chromophore Photoisomerization of a Fluorescent Protein Using Millisecond Synchrotron Serial Crystallography and Infrared Vibrational and Visible Spectroscopy

Abstract

The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm^-1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.

Figure 1

(a) Normalized UV/vis absorption spectra of rsEospa in the cis and trans states at acidic and neutral pH. At pH 5.5 the trans state was formed after illumination with 405 nm light, and at pH 8.4 the trans state was formed after illumination with 488 nm light. The anionic, neutral, and cationic assignments are discussed in the main text. The spectra are normalized at 280 nm. (b) Thermal recovery rates of the anionic-cis peak at 490 nm at pH 8.4 and 10 after full conversion with 488 nm light. (c) Absorption of the 490 and 390 nm peaks of unilluminated rsEospa as a function of pH. The solid lines follow the Henderson–Hasselbalch equation to fit the pK_a of 8.1.

Figure 2

(a–e) FTIR difference spectra for rsEospa and the wild-type Dronpa relative to the OFF state (dark-illuminated spectra). At pH/p²H 8.4 the sample was illuminated with 488 nm light; at pH/p²H 5.5 the sample was illuminated with 405 nm light. (f) Proposed schematic for switching above and below the pK_a. (g–j) 2F_o–F_c chromophore omit–density maps of the serial synchrotron crystallography structures for conditions: unilluminated pH 8.4, 488 nm illuminated at pH 8.4, unilluminated at pH 5.5, and 405 nm illuminated at pH 5.5.

Figure 3

Q-weighted difference electron density maps of different states of rsEospa plotted in red and blue at 3σ level. Shown in purple, gray, cyan, and yellow are the refined coordinates for pH 5.5 trans, pH 5.5 cis, pH 8.4 trans, and pH 8.4 cis, respectively. Difference maps are plotted for (a) pH 5.5 405 nm–pH 5.5 dark, (b) pH 8.4 488 nm–pH 8.4 dark, (c) pH 5.5 dark–pH 8.4 dark, and (d) pH 5.5 405 nm–pH 8.4 488 nm.

Figure 4

Chromophore alignment from the crystal structures of rsEospa at pH 5.5, pH 8.4 and Dronpa. The Dronpa structures were obtained from the Protein Data Bank (IDs: 2POX (trans) and 2IOV (cis)).