Rational Design of an Efficient, Genetically Encodable, Protein-Encased Singlet Oxygen Photosensitizer

Abstract

Singlet oxygen, O(2)(a(1)Δ(g)), plays a key role in many processes of cell signaling. Limitations in mechanistic studies of such processes are generally associated with the difficulty of controlling the amount and location of O(2)(a(1)Δ(g)) production in or on a cell. As such, there is great need for a system that (a) selectively produces O(2)(a(1)Δ(g)) in appreciable and accurately quantifiable yields and (b) can be localized in a specific place at the suborganelle level. A genetically encodable, protein-encased photosensitizer is one way to achieve this goal. Through a systematic and rational approach involving mutations to a LOV2 protein that binds the chromophore flavin mononucleotide (FMN), we have developed a promising photosensitizer that overcomes many of the problems that affect related systems currently in use. Specifically, by decreasing the extent of hydrogen bonding between FMN and a specific amino acid residue in the local protein environment, we decrease the susceptibility of FMN to undesired photoinitiated electron-transfer reactions that kinetically compete with O(2)(a(1)Δ(g)) production. As a consequence, our protein-encased FMN system produces O(2)(a(1)Δ(g)) with the uniquely large quantum efficiency of 0.25 ± 0.03. We have also quantified other key photophysical parameters that characterize this sensitizer system, including unprecedented H(2)O/D(2)O solvent isotope effects on the O(2)(a(1)Δ(g)) formation kinetics and yields. As such, our results facilitate future systematic developments in this field.

Solvent dependent photosensitized singlet oxygen production from an Ir(iii) complex: pointing to problems in studies of singlet-oxygen-mediated cell death

“…The data reported herein provide a nice justification for the design and development, for example, of genetically-encoded protein-encapsulated sensitizers. 19,71 In the ideal case under these latter conditions, the local environment of the sensitizer will remain the same and photophysical data …”

The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins

“…CALI with miniSOG was used to inhibit synaptic proteins [153,154], disrupt the CaMKII analogue UNC-43 in C. elegans [155], and in experiments to ablate cells [156]. SOPP, a newer variant engineered from miniSOG, is reported to have ~ 8-fold improved singlet oxygen quantum yield, but is yet to be tested in living cells [157]. A newly discovered LOV domain from Pseudomonas putida (Pp2FbFP L30M) is reported to have singlet oxygen quantum yield 3-fold higher than miniSOG [158], which may be a suitable template for future development of flavin-binding FPs as optogenetic tools to alter cellular function.…”

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

“…MiniSOG is a flavin mononucleotide (FMN) containing small protein investigated as a genetically encodable photosensitizer for the selective generation of singlet O 2 30, 31, 32. The bacterial NOX enzyme generates hydrogen peroxide (H 2 O 2 ) from O 2 by oxidizing NADH while the eukaryotic GOX naturally oxidizes glucose to H 2 O 2 and d ‐glucono‐δ‐lactone.…”