Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy

Abstract

Photosensitizing flavoproteins have great potential as tags for correlative light and electron microscopy (CLEM). We examine the photostability of miniSOG mutants and their ability to photo-oxidize diaminobenzidine, both key aspects for CLEM. Our experiments reveal a complex relation between these parameters and the production of different reactive oxygen species.

Tetramethylbenzidine: An Acoustogenic Photoacoustic Probe for Reactive Oxygen Species Detection

“…To this end, we added TMB to suspensions of wt DH10β E. coli bacteria as well as three genetically modified strains that express the ROS photosensitizing proteins miniSOG and its mutants Q103V and Q103L, respectively. These are flavin-binding proteins that produce 1 O 2 and other ROS in different proportions under exposure to blue light (460 nm) [ 66 , 67 , 68 ]. All strains produced a photoacoustic signal at 652 nm, likely due to light absorption by heme.…”

Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

“…Specifically, miniSOG is a singlet oxygen generator engineered from the flavin‐binding LOV2 domain that retains a modest fluorescence quantum yield . It was initially developed for the localized polymerization of diaminobenzidine for correlated electron microscopy (CLEM), and further studies have evaluated the efficacy of various mutants of miniSOG and their ability to produce various reactive oxygen species (ROS) . However, miniSOG and its variants suffer from photodegradation, which limits its use as a fluorescent tag for CLEM.…”

“…[68] It was initially developed for the localized polymerization of diaminobenzidine for correlated electron microscopy (CLEM), and further studies have evaluated the efficacy of various mutants of miniSOG and their ability to produce various reactive oxygen species (ROS). [69] However, miniSOG and its variants suffer from photodegradation, which limits its use as a fluorescent tag for CLEM. To circumvent this issue, tandem heterodimers of miniSOG and phiLOV2.1 were generated to combine the efficient ROS generation of miniSOG and the photostability of phiLOV2.1.…”

Photodynamic Physiology—Photonanomanipulations in Cellular Physiology with Protein Photosensitizers

“…The chromophore of Gln65-Tyr66-Gly67 (Takemoto et al, 2013) in SuperNova is highlighted in red. (B) The full amino acid sequence of miniSOG Q102V is from Rodríguez-Pulido et al (2016). The sequence is put into the protein structure website Swiss-model, three-dimensional model is then obtained after a build-model step.…”

“…This mutation reduces the hydrogen bond between Q102 and FMN, diminishing electron transfer, but enhancing energy transfer, with the net result of a much enhanced 1 O 2 quantum yield of 0.25 (Westberg et al, 2015, 2016). Another mutant, miniSOG Q102V (Figure 2), has an even higher 1 O 2 quantum yield of 0.39 (Rodríguez-Pulido et al, 2016). When miniSOG Q102L is expressed at the plasma membrane (by fusion with a PH domain) in C. elegans epithelial cells or in the cholinergic neurons, blue light illumination induces worm paralysis and neuronal injury, at efficiency higher than with miniSOG as the photosensitizer (Xu and Chisholm, 2016).…”