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Genetically encoded green-light-responsive photocaged lysine for sequential control of protein function.

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Researchers developed SCouK, a novel green-light-activatable unnatural amino acid (UAA) for precise protein control. This visible-light-responsive lysine derivative enables sequential photocontrol of protein activity in live cells, advancing biological studies.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Genetic code expansion enables site-specific incorporation of unnatural amino acids (UAAs) for protein control.
  • Existing photo-responsive UAAs are limited to UV/near-UV light, hindering simultaneous multi-protein regulation.
  • A need exists for visible-light-responsive UAAs to enable orthogonal and sequential protein manipulation.

Purpose of the Study:

  • To introduce a novel green-light-activatable lysine derivative, SCouK, for visible-light-mediated photocontrol of protein function.
  • To demonstrate the site-specific incorporation and photoactivation of SCouK in both bacterial and mammalian cells.
  • To showcase the sequential and orthogonal control of multiple proteins and biological pathways using SCouK.

Main Methods:

  • Genetic encoding of SCouK, a lysine derivative with a photolabile thiocoumarin moiety, into proteins.
  • Photoactivation of SCouK using visible light (green light, 520 nm) to restore protein function (EGFP, luciferase).
  • Demonstration of SCouK's application in controlling kinase activity and the cGAS-STING pathway, and sequential activation with a UV-light-activatable UAA.

Main Results:

  • SCouK was successfully genetically encoded and site-specifically incorporated into proteins in live bacterial and mammalian cells.
  • SCouK demonstrated efficient photodecaging by green light (520 nm) within 30 seconds, restoring protein functions.
  • SCouK enabled optical control of kinases, temporal interrogation of the cGAS-STING pathway, and, for the first time, sequential photoactivation of two distinct UAA-modified proteins.

Conclusions:

  • SCouK is the first visible-light-responsive lysine derivative for genetic encoding, offering precise, non-invasive protein manipulation.
  • Its green-light responsiveness, orthogonal activation, and high efficiency make it suitable for complex biological systems.
  • SCouK holds significant potential for advancing functional protein studies and therapeutic applications.