Picosecond fluorescence relaxation spectroscopy of the calcium-discharged photoproteins aequorin and obelin.
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.
van Oort
Bart
B
Laboratory of Biophysics, Wageningen University, The Netherlands.
Eremeeva
Elena V
EV
Koehorst
Rob B M
RB
Laptenok
Sergey P
SP
van Amerongen
Herbert
H
van Berkel
Willem J H
WJ
Malikova
Natalia P
NP
Markova
Svetlana V
SV
Vysotski
Eugene S
ES
Visser
Antonie J W G
AJ
Lee
John
J
D000331:Aequorin / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry*; D006207:Half-Life; D008164:Luminescent Proteins / Q000737:chemistry*; D008958:Models, Molecular; D013050:Spectrometry, Fluorescence / Q000379:methods*
D008164:Luminescent Proteins; C010497:obelin; D000331:Aequorin; D002118:Calcium