Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin

B.F. van Oort, E. Eremeeva, R.B.M. Koehorst, S. Laptenok, H. van Amerongen, W.J.H. van Berkel, N.P. Malikova, S.V. Markova, E.S. Vysotski, A.J.W.G. Visser, J. Lee

Research output: Contribution to journalArticleAcademicpeer-review

28 Citations (Scopus)

Abstract

Addition of calcium ions to the Ca2+-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (25000 cm-1) assigned as from the Ca2+-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t1/2 2 ps) in the case of the Ca2+-discharged obelin than aequorin (t1/2 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19400 cm-1, bound to Ca2+-discharged obelin, and 21300 cm-1, bound to Ca2+-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity
Original languageEnglish
Pages (from-to)10486-10491
JournalBiochemistry
Volume48
Issue number44
DOIs
Publication statusPublished - 2009

Keywords

  • ca2+-regulated photoproteins
  • violet bioluminescence
  • angstrom resolution
  • recombinant obelin
  • crystal-structure
  • w92f obelin
  • coelenterazine
  • mechanism
  • expression
  • proteins

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