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

Abstract

Addition of calcium ions to the Ca 2þ -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 (∼25 000 cm -1 ) assigned as from the Ca 2þ -discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t 1/2 ∼ 2 ps) in the case of the Ca 2þ -discharged obelin than aequorin (t 1/2 ∼ 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm -1 , bound to Ca 2þ -discharged obelin, and 21 300 cm -1 , bound to Ca 2þ -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.Bioluminescent animals are found in a variety of types occurring both terrestrially and in the ocean. More often than not, the chemistry of their light emission processes and the proteins involved are found quite unrelated. Well-studied cases, for example, are the bioluminescence of the firefly, which involves ATP and a substrate firefly luciferin, a benzthiozole derivative, and that of the photoprotein aequorin from the bioluminescent jellyfish Aequorea, which is triggered for light emission by Ca 2þ . Coelenterazine, an imidazopyrazinone derivative (Figure 1), is the luciferin (a generic term for the substrate) involved in many marine bioluminescent systems, including the ones subject to this present study, the Ca 2þ -regulated photoproteins, aequorin and obelin from the hydrazoan Obelia (1-3).A significant advance in uncovering the mechanism of bioluminescence from aequorin and obelin resulted from the determination of the high-resolution spatial structure of the two photoproteins (4-6). The coelenterazine was revealed residing in an internal cavity substituted with a peroxy group ( Figure 1B), as long suspected from earlier indirect evidence (7). Such a compound would be very unstable in free solution, but in the protein it appears to be frozen in place via a H-bond network to amino acid residues comprising the binding cavity. Model chemiluminescence studies with coelenterazine analogues had shown such a peroxide to be an intermediate, closing to a dioxetanone in the reaction pathway (8). The free energy produced by decarboxylation of this dioxetanone around 70 kcal/mol is sufficient to account for the energy of the photons of blue bioluminescence.Aequorin and obelin are EF-hand proteins belonging to the large family of Ca 2þ -binding proteins. They each contain three Ca 2þ -ligati...

Crystal structures of the F88Y obelin mutant before and after bioluminescence provide molecular insight into spectral tuning among hydromedusan photoproteins

“…If this pK* falls well below 6.5, which is the expected pK of His, rapid transient proton dissociation and its 'transient displacement' toward the N atom of His will occur, with simultaneous generation of the excited phenolate anion. As its fluorescence life- time is 5-6 ns [55], there is more than enough time for proton dissociation before radiation. However, in addition to the His residue, the p-OH of the phenol attached at C6 of coelenterazine is also surrounded by Trp and Tyr in aequorin and mitrocomin or Trp and Phe in obelin and clytin within hydrogen bonding distance.…”

Enzymatic Responses to Low-Intensity Radiation of Tritium

“…CLM-FPs are products of bioluminescent reactions of coelenterates that constitute a potential use in biomedicine as biomarkers [36][37][38]. The fluorescence characteristics of CLM-FPs were intensively studied [39][40][41][42][43][44][45][46]; they were found to be sensitive to external exposures: excitation energy [44], chemical agents [47,48], and radioactivity [49,50]. Recently, [51] suggested CLM-FPs for intracellular application as being the simplest toxicity bioassay.…”

Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study

“…The product of bioluminescent reaction of obelin, 'discharged obelin', CLM-containing protein, is under theoretical [9][10][11][12][13][14] and experimental [17,18] investigation now. Time-resolved fluorescence experiments were conducted in [23] to determine fluorescence characteristics of neutral and ionized forms of discharged obelin. Components of complex photoluminescent spectra of discharged obelin and aequorin were studied and attributed to the different forms of CLM [24]; changes of spectral composition were analyzed under variation of calcium concentration [25], exposure to higher temperature [26,27] and exogenous compounds [28][29][30].…”