title: Picosecond fluorescence relaxation spectroscopy of the calcium-discharged photoproteins aequorin and obelin.
abstract: Addition of calcium ions to the Ca(2+)-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 (approximately 25,000 cm(-1)) assigned as from the Ca(2+)-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t(1/2) approximately 2 ps) in the case of the Ca(2+)-discharged obelin than aequorin (t(1/2) approximately 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm(-1), bound to Ca(2+)-discharged obelin, and 21 300 cm(-1), bound to Ca(2+)-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.
authors.affiliation: Laboratory of Biophysics, Wageningen University, The Netherlands.
authors.forename: Bart
authors.initials: B
authors.lastname: van Oort
authors.forename: Elena V
authors.initials: EV
authors.lastname: Eremeeva
authors.forename: Rob B M
authors.initials: RB
authors.lastname: Koehorst
authors.forename: Sergey P
authors.initials: SP
authors.lastname: Laptenok
authors.forename: Herbert
authors.initials: H
authors.lastname: van Amerongen
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
authors.forename: Natalia P
authors.initials: NP
authors.lastname: Malikova
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Markova
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Vysotski
authors.forename: Antonie J W G
authors.initials: AJ
authors.lastname: Visser
authors.forename: John
authors.initials: J
authors.lastname: Lee
mesh_terms: D000331:Aequorin / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D006207:Half-Life; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; C010497:obelin; D000331:Aequorin; D002118:Calcium