Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein

Vladislav Verkhusha, N. A. Akovbian, E. N. Efremenko, S. D. Varfolomeyev, P. V. Vrzheshch

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state or the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.

Original languageEnglish (US)
Pages (from-to)1342-1351
Number of pages10
JournalBiochemistry (Moscow)
Volume66
Issue number12
DOIs
StatePublished - Dec 2001
Externally publishedYes

Fingerprint

Anthozoa
Denaturation
Fluorescence
Kinetics
Exponential functions
Recovery
Protons
Protein Denaturation
Proteins
red fluorescent protein
fluorescent protein 583
Protonation
Dichroism
Circular Dichroism
Monomers
Acids

Keywords

  • Chromophore
  • Denaturation
  • Kinetics
  • Maturation
  • Red fluorescent protein
  • Renaturation
  • Tetramer

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein. / Verkhusha, Vladislav; Akovbian, N. A.; Efremenko, E. N.; Varfolomeyev, S. D.; Vrzheshch, P. V.

In: Biochemistry (Moscow), Vol. 66, No. 12, 12.2001, p. 1342-1351.

Research output: Contribution to journalArticle

Verkhusha, Vladislav ; Akovbian, N. A. ; Efremenko, E. N. ; Varfolomeyev, S. D. ; Vrzheshch, P. V. / Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein. In: Biochemistry (Moscow). 2001 ; Vol. 66, No. 12. pp. 1342-1351.
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