Insight into the common mechanism of the chromophore formation in the red fluorescent proteins: The elusive blue intermediate revealed

Ksenia B. Bravaya, Oksana M. Subach, Nadezhda Korovina, Vladislav Verkhusha, Anna I. Krylov

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Understanding the chromophore maturation process in fluorescent proteins is important for the design of proteins with improved properties. Here, we present the results of electronic structure calculations identifying the nature of a blue intermediate, a key species in the process of the red chromophore formation in DsRed, TagRFP, fluorescent timers, and PAmCherry. The chromophore of the blue intermediate has a structure in which the π-system of the imidazole ring is extended by the acylimine bond, which can be represented by the model N-[(5-hydroxy-1H-imidazole-2yl)methylidene]acetamide (HIMA) compound. Ab initio and QM/MM calculations of the isolated model and protein-bound (mTagBFP) chromophores identify the anionic form of HIMA as the only structure that has absorption that is consistent with the experiment and is stable in the protein binding pocket. The anion and zwitterion are the only protonation forms of HIMA whose absorption (421 and 414 nm, or 2.95 and 3.00 eV) matches the experimental spectrum of the blue form in DsRed (the absorption maximum is 408 nm or 3.04 eV) and mTagBFP (400 nm or 3.10 eV). The QM/MM optimization of the protein-bound anionic form results in a structure that is close to the X-ray one, whereas the zwitterionic chromophore is unstable in the protein binding pocket and undergoes prompt proton transfer. The computed excitation energy of the protein-bound anionic form of the mTagBFP-like chromophore (3.04 eV) agrees with the experimental absorption spectrum of the protein. The DsRed-like chromophore formation in red fluorescent proteins is revisited on the basis of ab initio results and verified by directed mutagenesis revealing a key role of the amino acid residue 70, which is the second after the chromophore tripeptide, in the formation process.

Original languageEnglish (US)
Pages (from-to)2807-2814
Number of pages8
JournalJournal of the American Chemical Society
Volume134
Issue number5
DOIs
StatePublished - Feb 8 2012

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Chromophores
Proteins
Protein Binding
Mutagenesis
Proton transfer
Anions
red fluorescent protein
Protons
Excitation energy
Protonation
X-Rays
Electronic structure
Amino acids
Amino Acids
Absorption spectra
Negative ions
imidazole
X rays
fluorescent protein 583
acetamide

ASJC Scopus subject areas

  • Chemistry(all)
  • Catalysis
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Insight into the common mechanism of the chromophore formation in the red fluorescent proteins : The elusive blue intermediate revealed. / Bravaya, Ksenia B.; Subach, Oksana M.; Korovina, Nadezhda; Verkhusha, Vladislav; Krylov, Anna I.

In: Journal of the American Chemical Society, Vol. 134, No. 5, 08.02.2012, p. 2807-2814.

Research output: Contribution to journalArticle

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