A photoswitchable orange-to-far-red fluorescent protein, PSmOrange

Oksana M. Subach, George H. Patterson, Li Min Ting, Yarong Wang, John S. Condeelis, Vladislav V. Verkhusha

Research output: Contribution to journalArticle

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Abstract

We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a new chromophore containing N-acylimine with a co-planar carbon-oxygen double bond.

Original languageEnglish (US)
Pages (from-to)771-780
Number of pages10
JournalNature Methods
Volume8
Issue number9
DOIs
StatePublished - Sep 1 2011

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ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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