Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed

Abstract

KillerRed is the only known fluorescent protein that demonstrates notable phototoxicity, exceeding that of the other green and red fluorescent proteins by at least 1,000-fold. KillerRed could serve as an instrument to inactivate target proteins or to kill cell populations in photodynamic therapy. However, the nature of KillerRed phototoxicity has remained unclear, impeding the development of more phototoxic variants. Here we present the results of a high resolution crystallographic study of KillerRed in the active fluorescent and in the photobleached non-fluorescent states. A unique and striking feature of the structure is a waterfilled channel reaching the chromophore area from the end cap of the ␤-barrel that is probably one of the key structural features responsible for phototoxicity. A study of the structure-function relationship of KillerRed, supported by structure-based, site-directed mutagenesis, has also revealed the key residues most likely responsible for the phototoxic effect. In particular, Glu 68 and Ser 119, located adjacent to the chromophore, have been assigned as the primary trigger of the reaction chain. The green fluorescent protein (GFP)2 and related proteins have become efficient noninvasive tools in cell biology and biomedicine for visualizing and monitoring the internal processes within cells or whole organisms (1-8). The multicolor labeling technologies, based on fluorescent proteins (FPs), have found important biomedical applications in the studies of various aspects of cancer, including primary tumor growth, tumor cell motility and invasion, metastatic seeding, colonization, and angiogenesis (9 -11).The recent development of the first genetically encoded photosensitizer, KillerRed (SWISS-PROT/TrEMBL data base sequence ID Q2TCH5), a highly phototoxic red fluorescent protein (12, 13), opened a new area of FP application. KillerRed is a red fluorescent protein characterized by excitation and emission maxima at 585 and 610 nm, respectively. This genetic variant was engineered from non-fluorescent and non-phototoxic chromoprotein anm2CP from Hydrozoa jellyfish (sequence ID Q6RYS4). Upon irradiation by green light at the wavelength of 520 -590 nm, KillerRed generates the reactive oxygen species (ROS), accompanied by profound self photobleaching. The ROS-induced phototoxicity of KillerRed is at least 3 orders of magnitude higher than that of other fluorescent proteins exhibiting low background phototoxicity (12). Such a unique property of KillerRed could find use in light-induced inactivation of target proteins and in precise cell killing. Unlike chemical photosensitizers, KillerRed can be directly expressed by a target cell, both individually and in fusion with a target protein. The most exciting future application of KillerRed may be in photodynamic therapy of cancer. This phototoxic agent, precisely delivered to solid tumors by a viral vector, could serve as an intrinsically generated photosensitizer, causing light-induced tumor destruction. Therefore, understanding the relationship between...

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

“…[9,10] The presence of a long water-filled channel that connects the chromophore with the external solvent, which facilitates the diffusion of molecular oxygen, is also crucial in the properties of KillerRed. [9,11] Another important ROS such as singlet oxygen ( 1 O 2 ) has also been detected from KillerRed, though to a lesser extent, [8] and it does not play a major role in the KillerRed CALI effect. [10] 1 O 2 , however, seems to be the main ROS that participates in CALI when using other FPs such as the enhanced (E)GFP.…”

Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery

“…This approach has been of limited value because targeting proteins of interest mostly relied on microinjection of fluorophore-conjugated antibodies (Surrey et al, 1998;Jay and Sakurai, 1999). However, the development of Killer Red, a derivative of the Hydrazoa jellyfish chromoprotein anm2CP that generates at least 1000-fold increase in reactive oxygen species over green fluorescence protein (GFP), presents the possibility of simply expressing Killer Red fusion proteins (Bulina et al, 2006;Carpentier et al, 2009;Pletnev et al, 2009;Serebrovskaya et al, 2009;Destaing et al, 2010;Teh et al, 2010).…”

Targeted Photodynamic Virotherapy Armed with a Genetically Encoded Photosensitizer

“…Previous reports have suggested that light-mediated KillerRed activation induces ROS generation (3,5) and apoptosis (5,7) and these data are consistent with our results. Analysis of KillerRed structure by an X-ray (19) and a high-resolution crystallographic study (20) revealed that a waterfilled channel that reacts with the chromophore is responsible for the generation of ROS, which is mainly composed of singlet oxygen (3,21). The intracellular localization of KillerRed that generates ROS is an especially critical factor for KillerRed-mediated cell cytotoxicity.…”