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Related Experiment Videos

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.

Nature Biotechnology
|November 20, 2003
PubMed
Summary

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.

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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.

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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.