Tailing miniSOG: structural bases of the complex photophysics of a flavin-binding singlet oxygen photosensitizing protein

Abstract

miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.

Photoinduced damage of AsLOV2 domain is accompanied by increased singlet oxygen production due to flavin dissociation

“…A higher number of oxidative modifications of amino acids detected in our work in comparison with a very recent paper by Torra et al 29 can be attributed to the differences in the solution irradiation (wavelength, power, etc.) and partially also to the higher sensitivity and resolution obtained by our LC-MS/MS analysis on FT-ICR MS and combination of proteomics approaches directed at localization of the modification sites.…”

“…Our approach truly analyzes all protein states present in the sample and, in addition, the top-down analysis allows the identification of the first-hit residues. In contrast, Torra et al 29 identified the oxidized residues in the crystal structure, hence the possibility that they observed just the crystallization-capable population cannot be excluded.…”

“…This finding was explained as the result of progressive photoinactivation of certain amino acids, such as His, Met, Phe, Trp, and Tyr, that are responsible for 1 O 2 quenching 5,8,28 and/or the buildup of FMN photoproducts 21 . Indeed, very recently Torra et al 29 showed that the irradiation of miniSOG leads not only to oxidation of several residues, which are possible electron donors to FMN, but also to phototransformation of FMN to lumichrome, which results in facilitation of the access of molecular oxygen to the isoalloxazine ring of FMN.…”

Permanent Photodynamic Activation of the Cholecystokinin 2 Receptor

“…Other than CCK2R sensitization through SALPC photodynamic action, we also examined photodynamic CCK2R modulation with the genetically encoded protein photosensitizer miniSOG. miniSOG (106 residues) with a noncovalently bound fluorophore flavin mononucleotide (FMN) is a protein photosensitizer with sufficient 1 O 2 quantum yield (≥0.03) [53,54]. Other than generating 1 O 2 , miniSOG, upon excitation with blue light (448 nm), also emits green (500 nm) fluorescence [55,56] and therefore can be used as a fluorescent tag for protein subcellular localization and protein-protein interactions [57][58][59][60][61].…”

Engineering Glowing Chemogenetic Hybrids for Spying on Cells

“…Crystallographic studies showed an increase in rigidity of the chromophore's environment compared to wild-type LOV2 suggesting that oxygen diffusion can occur properly. [84] When irradiated with blue light, miniSOG fluoresces and generates both superoxide anion (and its derivatives) and singlet oxygen ( 1 O 2 ) through type I and II mechanisms. [85,86] This dual function allowed the imaging of MiniSOG by correlative light and electron microscopy (CLEM) in Caenorhabditis elegans and mice synaptic cells.…”