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

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