Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer

Abstract

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (¹ O₂ ). However, its ¹ O₂ production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase ¹ O₂ production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective ¹ O₂ production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low ¹ O₂ production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

Keywords: LOV2 domain; flavin cofactor; genetically encoded photosensitizers; miniSOG; singlet oxygen.

FIGURE 1

(a) Crystal structure of LOV2 domain of Avena sativa (AsLOV2; PDB: 2v0u). The N‐terminal turn‐helix‐turn motif is shown in dark blue, and the Jα helix is shown in red. (b) Detailed view of the binding site of the isoalloxazine moiety, indicating a tight arrangement around the isoalloxazine ring. (c) Isoalloxazine ring‐binding site, highlighting the five amino acids that may be replaced based on the selection criteria (see main text). The amino acids Val416 and Thr418 were modified in this study.

FIGURE 2

(a) Free energy profiles of flavin mononucleotide (FMN) binding to wild type and mutant LOV2 domain of Avena sativa (AsLOV2) domains are shown as a function of the position of the center of FMN. The origin of the z‐coordinate (z _FMN = 0) corresponds to the center of the AsLOV2 domain (Figure S1). Snapshots at z _FMN = 7, 11, 15, 19, and 23 Å are shown. The position of FMN in the crystal structure (PDB ID = 2v0u) corresponds to z _FMN ≈7.8 Å. (b) and (c) far‐ and near‐UV circular dichroism spectra of the AsLOV2 variants expressed as the mean residue ellipticity and molar ellipticity, respectively. (d) Thermal denaturation of AsLOV2 monitored by FMN fluorescence. The symbols and lines correspond to experimental data and fits according to Equation (1), respectively. (e) DSC thermograms of AsLOV2 variants. AsLOV2 variants are colored as follows: WT (black), V416C (blue), T418C (red), and V416C/T418C (green) (left box).

FIGURE 3

Upper row—time‐resolved singlet oxygen phosphorescence of the LOV2 domain of Avena sativa (AsLOV2) variants, with the same scaled y‐axis for all plots. The color scheme represent the following accumulated incident energy: black open squares 0.2 J, red open circles 0.8 J, blue triangles 1.4 J, green reverse triangles 2 J, purple diamonds 2.6 J, and yellow ochre left triangles 3.2 J. The black lines correspond to fits by Equation (6). Lower row—fluorescence spectra of the AsLOV2 variants before irradiation (red curves), free flavin mononucleotide (green curves), and AsLOV2 variants after irradiation (black dotes). The blue line is the linear combination of the red and green curves. ¹O₂, singlet oxygen.

FIGURE 4

(a) Variation of amplitude [A _out] derived from Equation (6) as a function of time, reflecting the singlet oxygen phosphorescence of each LOV2 domain of Avena sativa (AsLOV2) variant shown in Figure 3 (upper row): wt (black squares), V416C (blue triangles), T418C (red circles), and V416C/T418C (green reverse triangles). (b) Correlation between the amplitude [A _out] derived from Equation (6) after the last round of irradiation and flavin mononucleotide (FMN) dissociation, as calculated from Figure 3. The correlation is described by linear equation y = 2.06x + 22.12 with R ² = 0.998. (c) Fluorescence intensity of the irradiation‐released FMN of the corresponding AsLOV2 after filtration experiment. No fluorescence was present in the filtrates of AsLOV2 wt and its variants before irradiation.

FIGURE 5

(a) The results of the extent of oxidation of selected b‐ and y‐fragment ions of irradiated samples are expressed as the mean ± standard deviation (SD) of three independent measurements. The inset in the y‐ions plot indicates the extent of doubly oxidized y127 and y148 fragment ions. (b) The sequence of wild type LOV2 domain of Avena sativa (AsLOV2) protein with the denoted fragment ions is displayed at the top of the figure. The His‐tag, which is not part of the AsLOV2 sequence, is colored in gray and underlined in red. The positions of the mutations V416C and T418C are shown in blue and red squares, respectively.