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LED-Based In Vitro Screening for Assessing Photoactivable Molecules in Bacterial Photodynamic Inactivation
Published on: January 24, 2025
Genetically targeted chromophore-assisted light inactivation
Oded Tour1, Rene M Meijer, David A Zacharias
1Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA.
Abstract:
Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochemically generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and alpha1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by approximately 90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or alpha1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected molecular specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen respectively inhibit or enhance CALI.
Insights
Researchers developed a new method for targeted protein inactivation using genetically encoded tags and a specific dye. This technique, ReAsH-based chromophore-assisted light inactivation (CALI), allows precise protein disabling in living cells without invasive procedures.
Area of Science:
- Cell biology
- Biochemistry
- Molecular genetics
Background:
- Protein function studies require precise methods for inactivation in living cells.
- Current chromophore-assisted light inactivation (CALI) methods often necessitate microinjection and antibodies, limiting their application.
Purpose of the Study:
- To develop a noninvasive, genetically targeted CALI method for protein inactivation.
- To demonstrate the efficacy and specificity of ReAsH-based CALI for connexin43 (Cx43) and alpha1C L-type calcium channels.
Main Methods:
- Genetically engineered cells expressing proteins tagged with tetracysteine (TC) motifs.
- Application of the membrane-permeant red biarsenical dye, ReAsH.
- Inactivation of target proteins using widefield illumination and assessment of singlet oxygen involvement.
Main Results:
- ReAsH-based CALI achieved approximately 90% inactivation of Cx43 and alpha1C channels within 30 seconds of illumination.
- The method is genetically targeted, eliminating the need for antibodies or microinjection.
- Control experiments with GFP-tagged proteins showed minimal effects, confirming the specificity of ReAsH-CALI.
- ReAsH-mediated CALI primarily involves singlet oxygen, as indicated by the effects of quenchers and enhancers.
Conclusions:
- ReAsH-based CALI provides a powerful, noninvasive tool for studying protein function with high spatial and temporal control.
- This genetically targeted approach overcomes limitations of previous CALI methods.
- The mechanism of ReAsH-CALI involves singlet oxygen, offering insights into its photochemical activity.
