Orange fluorescent proteins: Structural studies of LSSmOrange, PSmOrange and PSmOrange2

Sergei Pletnev, Daria Shcherbakova, Oksana M. Subach, Nadya V. Pletneva, Vladimir N. Malashkevich, Steven C. Almo, Zbigniew Dauter, Vladislav Verkhusha

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

A structural analysis of the recently developed orange fluorescent proteins with novel phenotypes, LSSmOrange (λexem at 437/572 nm), PSmOrange (λexem at 548/565 nm and for photoconverted form at 636/662 nm) and PSmOrange2 (λexem at 546/561 nm and for photoconverted form at 619/651 nm), is presented. The obtained crystallographic structures provide an understanding of how the ensemble of a few key mutations enabled special properties of the orange FPs. While only a single Ile161Asp mutation, enabling excited state proton transfer, is critical for LSSmOrange, other substitutions provide refinement of its special properties and an exceptional 120 nm large Stokes shift. Similarly, a single Gln64Leu mutation was sufficient to cause structural changes resulting in photoswitchability of PSmOrange, and only one additional substitution (Phe65Ile), yielding PSmOrange2, was enough to greatly decrease the energy of photoconversion and increase its efficiency of photoswitching. Fluorescence of photoconverted PSmOrange and PSmOrange2 demonstrated an unexpected bathochromic shift relative to the fluorescence of classic red FPs, such as DsRed, eqFP578 and zFP574. The structural changes associated with this fluorescence shift are of considerable value for the design of advanced far-red FPs. For this reason the chromophore transformations accompanying photoconversion of the orange FPs are discussed.

Original languageEnglish (US)
Article numbere99136
JournalPLoS One
Volume9
Issue number6
DOIs
StatePublished - Jun 24 2014

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Fluorescence
fluorescence
mutation
Mutation
Substitution reactions
Proteins
Proton transfer
Chromophores
Excited states
Structural analysis
protons
Protons
Phenotype
phenotype
energy
fluorescent proteins
fluorescent protein 583

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Orange fluorescent proteins : Structural studies of LSSmOrange, PSmOrange and PSmOrange2. / Pletnev, Sergei; Shcherbakova, Daria; Subach, Oksana M.; Pletneva, Nadya V.; Malashkevich, Vladimir N.; Almo, Steven C.; Dauter, Zbigniew; Verkhusha, Vladislav.

In: PLoS One, Vol. 9, No. 6, e99136, 24.06.2014.

Research output: Contribution to journalArticle

Pletnev, Sergei ; Shcherbakova, Daria ; Subach, Oksana M. ; Pletneva, Nadya V. ; Malashkevich, Vladimir N. ; Almo, Steven C. ; Dauter, Zbigniew ; Verkhusha, Vladislav. / Orange fluorescent proteins : Structural studies of LSSmOrange, PSmOrange and PSmOrange2. In: PLoS One. 2014 ; Vol. 9, No. 6.
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