The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein

Jennifer L. Watkins, Hanseong Kim, Michele L. Markwardt, Liqing Chen, Raimund Fromme, Mark A. Rizzo, Rebekka Wachter

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Genetically encoded cyan fluorescent proteins (CFPs) bearing a tryptophan-derived chromophore are commonly used as energy-donor probes in Förster resonance energy transfer (FRET) experiments useful in live cell-imaging applications. In recent years, significant effort has been expended on eliminating the structural and excited-state heterogeneity of these proteins, which has been linked to undesirable photophysical properties. Recently, mCerulean3, a descendant of enhanced CFP, was introduced as an optimized FRET donor protein with a superior quantum yield of 0.87. Here, the 1.6 Å resolution X-ray structure of mCerulean3 is reported. The chromophore is shown to adopt a planar trans configuration at low pH values, indicating that the acid-induced isomerization of Cerulean has been eliminated. β-Strand 7 appears to be well ordered in a single conformation, indicating a loss of conformational heterogeneity in the vicinity of the chromophore. Although the side chains of Ile146 and Leu167 appear to exist in two rotamer states, they are found to be well packed against the indole group of the chromophore. The Ser65 reversion mutation allows improved side-chain packing of Leu220. A structural comparison with mTurquoise2 is presented and additional engineering strategies are discussed.

Original languageEnglish (US)
Pages (from-to)767-773
Number of pages7
JournalActa Crystallographica Section D: Biological Crystallography
Volume69
Issue number5
DOIs
StatePublished - May 2013

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Energy Transfer
Tryptophan
Proteins
X-Rays
Mutation
Acids
Cyan Fluorescent Protein
indole
enhanced cyan fluorescent protein

Keywords

  • chromophores
  • directed evolution
  • ECFP
  • enhanced cyan fluorescent proteins
  • FRET
  • GFP-like proteins
  • quantum yield
  • structure-guided protein engineering

ASJC Scopus subject areas

  • Structural Biology

Cite this

The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein. / Watkins, Jennifer L.; Kim, Hanseong; Markwardt, Michele L.; Chen, Liqing; Fromme, Raimund; Rizzo, Mark A.; Wachter, Rebekka.

In: Acta Crystallographica Section D: Biological Crystallography, Vol. 69, No. 5, 05.2013, p. 767-773.

Research output: Contribution to journalArticle

Watkins, Jennifer L. ; Kim, Hanseong ; Markwardt, Michele L. ; Chen, Liqing ; Fromme, Raimund ; Rizzo, Mark A. ; Wachter, Rebekka. / The 1.6 Å resolution structure of a FRET-optimized Cerulean fluorescent protein. In: Acta Crystallographica Section D: Biological Crystallography. 2013 ; Vol. 69, No. 5. pp. 767-773.
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