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Efficient switching of mCherry fluorescence using chemical caging.

Bas M C Cloin1, Elke De Zitter2, Desiree Salas1

  • 1Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3584 CH Utrecht, The Netherlands.

Proceedings of the National Academy of Sciences of the United States of America
|June 21, 2017
PubMed
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Chemically switching the red fluorescent protein mCherry to a blue state using beta-mercaptoethanol (βME) enables new imaging techniques. This reversible process allows for advanced microscopy on existing biological constructs without new labels.

Area of Science:

  • Biochemistry
  • Microscopy
  • Structural Biology

Background:

  • Dynamic fluorophores are crucial for advanced imaging techniques like superresolution microscopy.
  • Developing new fluorescent probes often requires introducing novel labels that may interfere with biological constructs.

Purpose of the Study:

  • To investigate a method for chemically inducing a reversible blue fluorescence state in the red fluorescent protein mCherry.
  • To demonstrate the applicability of this chemically induced fluorescence switching for single-molecule localization microscopy (SMLM).

Main Methods:

  • Incubation of mCherry with beta-mercaptoethanol (βME) to induce blue fluorescence.
  • Recovery of red fluorescence by washing out βME or via violet light irradiation.
  • Mechanism investigation using X-ray crystallography, NMR spectroscopy, and quantum-mechanical calculations.
Keywords:
fluorescent proteinslocalization microscopymCherryphotoactivationβ-mercaptoethanol

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Main Results:

  • mCherry can be chemically switched to a blue-fluorescent state by βME, with up to 80% recovery to the red state.
  • This chemically induced switching is applicable to single-molecule localization microscopy (SMLM) in cells expressing mCherry.
  • The βME-induced quenching involves direct addition to and reduction of the chromophore.

Conclusions:

  • This chemical switching strategy expands SMLM imaging capabilities to a wider range of existing biological models.
  • The study provides novel insights into the chemical mechanisms governing the dynamics of fluorescent proteins.