Green monomeric photosensitizing fluorescent protein for photo-inducible protein inactivation and cell ablation

Abstract

BackgroundPhotosensitizing fluorescent proteins, which generate reactive oxygen species (ROS) upon light irradiation, are useful for spatiotemporal protein inactivation and cell ablation. They give us clues about protein function, intracellular signaling pathways and intercellular interactions. Since ROS generation of a photosensitizer is specifically controlled by certain excitation wavelengths, utilizing colour variants of photosensitizing protein would allow multi-spatiotemporal control of inactivation. To expand the colour palette of photosensitizing protein, here we developed SuperNova Green from its red predecessor, SuperNova.ResultsSuperNova Green is able to produce ROS spatiotemporally upon blue light irradiation. Based on protein characterization, SuperNova Green produces insignificant amounts of singlet oxygen and predominantly produces superoxide and its derivatives. We utilized SuperNova Green to specifically inactivate the pleckstrin homology domain of phospholipase C-δ1 and to ablate cancer cells in vitro. As a proof of concept for multi-spatiotemporal control of inactivation, we demonstrate that SuperNova Green can be used with its red variant, SuperNova, to perform independent protein inactivation or cell ablation studies in a spatiotemporal manner by selective light irradiation.ConclusionDevelopment of SuperNova Green has expanded the photosensitizing protein toolbox to optogenetically control protein inactivation and cell ablation.Electronic supplementary materialThe online version of this article (10.1186/s12915-018-0514-7) contains supplementary material, which is available to authorized users.

Photodynamic Activation of Cholecystokinin 1 Receptor with Different Genetically Encoded Protein Photosensitizers and from Varied Subcellular Sites

“…For possible future in vivo applications, KillerRed might be better-suited due to its excitation by red light, instead of by blue light for miniSOG, miniSOG2, SOPP, Mr4511 C71G , and DsFbFP. It would be ideal if KillerRed could be subjected to further annotations, to shift its maximal excitation peak toward even longer wavelengths (a red shift), possibly by genetic code expansion [ 73 ], instead of the blue shifts observed in KillerOrange [ 46 , 47 ] and GreenSuperNova [ 45 ]. Although KillerRed is twice the size of miniSOG and larger than MR4511 C71G , DsFbFP (see Table A2 ), KillerRed fusion to CCK1R (CCK1R-KillerRed) were found to still result in, after light irradiation, the effective photodynamic activation of the in-frame CCK1R [ 31 ].…”

“…Different variants of KillerRed and miniSOG have also appeared, either to monomerize the KillerRed dimer (SuperNova and GreenSuperNova) [ 44 , 45 ], to blue-shift the KillerRed excitation peak (GreenSuperNova and KillerOrange) [ 45 , 46 , 47 ], or for graded increases in miniSOG 1 O 2 quantum yields (miniSOG2; miniSOG Q103L ; or singlet oxygen protein photosensitizer—SOPP, SOPP2, or SOPP3) [ 38 , 48 , 49 , 50 , 51 , 52 ], for enhanced miniSOG photostability after tethered dimerization with a more stable monomer (phiSOG and phiSOG Q103V ) [ 53 ]. A red fluorescent protein (TagRFP) for tagging larger proteins has been found to show a noted 1 O 2 quantum yield [ 54 ].…”

Optical manipulation of molecular function by chromophore-assisted light inactivation

“… 43 ) In addition, a mutant SuperNova-Green with maximum absorption at 440 nm was also developed by introducing a mutation containing Y66W and V44A in and around its chromophore. 44 ) This is expected to make it possible to multitask the CALI method, where multiple molecules can be manipulated with different wavelengths in the living cell. Because SuperNova has many advantages compared with KillerRed, as described above, it is gradually being applied for in vitro and in vivo CALI experiments (Tables 2 and 3 ).…”

“…To date, only one-color variants have been reported for SuperNova and KillerRed. 43 , 44 ) In the future, it is desired to establish a multicolored technology, similar to GFP, which can manipulate the behavior of many molecules simultaneously.…”

Molecular Tools to Generate Reactive Oxygen Species in Biological Systems

“…KillerRed is believed to mainly generate O2 •− via the type I reaction. [47][48][49] Recent effort has resulted in its monomeric and/or color-shifted variants, such as SuperNova, 50 SuperNova Green, 51 KillerOrange, 52 and mKillerOrange. 53 In addition to GFP analogs, some flavoproteins are also effective photosensitizers.…”