Photoswitchable red fluorescent protein with a large Stokes shift

Kiryl D. Piatkevich, Brian P. English, Vladimir N. Malashkevich, Hui Xiao, Steven C. Almo, Robert H. Singer, Vladislav V. Verkhusha

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

Original languageEnglish (US)
Pages (from-to)1402-1414
Number of pages13
JournalChemistry and Biology
Volume21
Issue number10
DOIs
StatePublished - Oct 23 2014

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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