Dynamic fluorescence properties of the bacterial luciferase intermediates

Abstract

Three fluorescent species produced by the reaction of bacterial luciferase from Vibrio harveyi with its substrates have the same dynamic fluorescence properties, namely, a dominant fluorescence decay of lifetime of 10 ns and a rotational correlation time of 100 ns at 2 degrees C. These three species are the metastable intermediate formed with the two substrates FMNH2 and O2, both in its low-fluorescence form and in its high-fluorescence form following light irradiation, and the fluorescent transient formed on including the final substrate tetradecanal. For native luciferase, the rotational correlation time is 62 or 74 ns (2 degrees C) derived from the decay of the anisotropy of the intrinsic fluorescence at 340 nm or the fluorescence of bound 8-anilino-1-naphthalenesulfonic acid (470 nm), respectively. The steady-state anisotropy of the fluorescent intermediates is 0.34, and the fundamental anisotropy from a Perrin plot is 0.385. The high-fluorescence intermediate has a fluorescence maximum at 500 nm, and its emission spectrum is distinct from the bioluminescence spectrum. The fluorescence quantum yield is 0.3 but decreases on dilution with a quadratic dependence on protein concentration. This, and the large value of the rotational correlation time, would be explained by protein complex formation in the fluorescent intermediate states, but no increase in protein molecular weight is observed by gel filtration or ultracentrifugation. The results instead favor a proposal that, in these intermediate states, the luciferase undergoes a conformational change in which its axial ratio increases by 50%.

Properties of recombinant fluorescent proteins from Photobacterium leiognathi and their interaction with luciferase intermediates

“…Emission decay measurements were made with laser system and single-photon counting Petushkov et al electronics described in detail elsewhere (Lee et al, 1989b). The method of data collection and analysis was as adequately detailed elsewhere (Lee et al, 1988(Lee et al, , 1989a(Lee et al, ,b, 1991b, except with a few modifications. The sample was contained in a 3 mL thermostated cuvette, and the path lengths for excitation and emission were 10 mm.…”

Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature.

“…It can be shown that for a spherical body measured at 2°C, 4 (in units of ns) is roughly equal to the molecular mass of the protein (in kDa). Dynamic fluorescence measurements of ANS bound to the wild-type luciferase and the fusion proteins in peak I and II were performed by laser excitation and single photon counting electronics (18).…”

Bioluminescence spectral and fluorescence dynamics study of the interaction of lumazine protein with the intermediates of bacterial luciferase bioluminescence

“…' The absorption spectrum of I1 is typical of a 4a,5-dihydroflavin, and it is weakly fluorescent, with an emission maximum around 500 nm (Hastings et al, 1973;Lee et al, 1988). The fluorescence quantum +Supported by grants from the National Science Foundation (DMB85-12361 and PCM 83-12669) and the National Institutes of Health (GM-28139, RR-02015, and RR-02389).…”

“…The fluorescence quantum +Supported by grants from the National Science Foundation (DMB85-12361 and PCM 83-12669) and the National Institutes of Health (GM-28139, RR-02015, and RR-02389). yield is increased severalfold to 0.3 by irradiation with light Tu, 1979Tu, , 1986Lee et al, 1988).…”