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 water-filled channel reaching the chromophore area from the end cap of the beta-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.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2797274 | PMC |
| http://dx.doi.org/10.1074/jbc.M109.054973 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
OSP-1 protects neurons from autophagic cell death induced by acute oxidative stress.
Nat Commun
January 2025
Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, QLD, Australia.
Oxidative stress, caused by the accumulation of reactive oxygen species (ROS), is a pathological factor in several incurable neurodegenerative conditions as well as in stroke. However, our knowledge of the genetic elements that can be manipulated to protect neurons from oxidative stress-induced cell death is still very limited. Here, using Caenorhabditis elegans as a model system, combined with the optogenetic tool KillerRed to spatially and temporally control ROS generation, we identify a previously uncharacterized gene, oxidative stress protective 1 (osp-1), that protects C.
View Article and Find Full Text PDFUnraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer.
Biomedicines
November 2024
Institute of Experimental Oncology and Biomedical Technologies, Privolzhsky Research Medical University, Minin and Pozharsky Square, 10/1, 603005 Nizhny Novgorod, Russia.
Background: Despite the fundamental importance of cell membrane microviscosity, changes in this biophysical parameter of membranes during photodynamic therapy (PDT) have not been fully understood.
Methods: In this work, changes in the microviscosity of membranes of live HeLa Kyoto tumor cells were studied during PDT with KillerRed, a genetically encoded photosensitizer, in different cellular localizations. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive BODIPY2 rotor.
Chromophore-Assisted Light Inactivation for Protein Degradation and Its Application in Biomedicine.
Bioengineering (Basel)
June 2024
State Key Laboratory of Targeting Oncology, National Center for International Research of Bio-Targeting Theranostics, Guangxi Key Laboratory of Bio-Targeting Theranostics, Collaborative Innovation Center for Targeting Tumor Diagnosis and Therapy, Guangxi Medical University, Nanning 530021, China.
The functional investigation of proteins holds immense significance in unraveling physiological and pathological mechanisms of organisms as well as advancing the development of novel pharmaceuticals in biomedicine. However, the study of cellular protein function using conventional genetic manipulation methods may yield unpredictable outcomes and erroneous conclusions. Therefore, precise modulation of protein activity within cells holds immense significance in the realm of biomedical research.
View Article and Find Full Text PDFUpconversion dual-photosensitizer-expressing bacteria for near-infrared monochromatically excitable synergistic phototherapy.
Sci Adv
March 2024
Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Institute of Molecular Medicine, State Key Laboratory of Systems Medicine for Cancer, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
Synergistic phototherapy stands for superior treatment prospects than a single phototherapeutic modality. However, the combined photosensitizers often suffer from incompatible excitation mode, limited irradiation penetration depth, and lack of specificity. We describe the development of upconversion dual-photosensitizer-expressing bacteria (UDPB) for near-infrared monochromatically excitable combination phototherapy.
View Article and Find Full Text PDFEffects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.
Int J Mol Sci
January 2024
Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, Malaya Pirogovskaya, 1a, 119435 Moscow, Russia.
This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!