DIRECT MEASUREMENT OF EXCITATION TRANSFER IN THE PROTEIN COMPLEX OF BACTERIAL LUCIFERASE HYDROXYFLAVIN AND THE ASSOCIATED YELLOW FLUORESCENCE PROTEINS FROM VIBRIO-FISCHERI Y1
Vn. Petushkov et al., DIRECT MEASUREMENT OF EXCITATION TRANSFER IN THE PROTEIN COMPLEX OF BACTERIAL LUCIFERASE HYDROXYFLAVIN AND THE ASSOCIATED YELLOW FLUORESCENCE PROTEINS FROM VIBRIO-FISCHERI Y1, Biochemistry, 35(25), 1996, pp. 8413-8418
Time-resolved fluorescence was used to directly measure the energy tra
nsfer rate constant in the protein-protein complex involved in the yel
low bioluminescence of Vibrio fischeri, strain Y1. In this reaction th
e putative donor is the fluorescent transient intermediate, luciferase
hydroxyflavin, which exhibits a major fluorescence lifetime of the bo
und flavin of 10 ns. On addition of the acceptor, the V. fischeri yell
ow fluorescence protein containing either FMN or riboflavin as ligand,
a rapid decay time, 0.25 ns, becomes predominant. The same results ar
e observed using rec-luciferase from Photobacterium leiognathi to prod
uce the donor. Because of favorable spectral separation in this system
, this rapid decay rate of 4 ns(-1), can be directly equated to the en
ergy transfer rate. This rate is ten times higher than the rate previo
usly observed in the Photobacterium luciferase hydroxyflavin-lumazine
protein, donor-acceptor system, derived from emission anisotropy measu
rements. This ten-times ratio is close to the ratio of spectral overla
ps of the donor fluorescence with the acceptor absorption, between the
se two systems, so it is concluded that the topology of the protein co
mplexes in both cases, must be very similar. Energy transfer is also m
onitored by the loss of steady-state fluorescence intensity at 460 nm
of the donor, on addition of the acceptor protein. A fluorescence titr
ation indicates that luciferase hydroxyflavin and the yellow protein c
omplex with a 1:1 stoichiometry with a K-d of 0.7 mu M (0 degrees C).
These parameters account for the bioluminescence spectral shifting eff
ects observed in these reactions.