Time-resolved fluorescence study of the dissociation of FMN from the yellow fluorescence protein from Vibrio fischeri

Time-resolved fluorescence spectroscopy of the flavin mononucleotide (FMN) prosthetic group of the yellow fluorescence protein (YFP) from Vibrio fischeri has provided quantitative, thermodynamic information on the FMN-apoYFP equilibrium in aqueous buffer, In diluted aqueous solution two fluorescent species could be identified by distinct fluorescence lifetimes and rotational correlation times originating from free- and protein-bound PMN. Quantitation of the amounts of free and bound FMN in progressively larger dilutions of YFP in aqueous buffer yielded a dissociation constant of 0.40 mu M for the FMN-apoprotein complex at 20 degrees C, The single fluorescence Lifetime of YFP-bound FMN is very long (7.6 ns at 20 degrees C), suggesting a binding environment in which maximal emission is provided commensurate with its function as a bioluminescent emitter, The single correlation time of 14.8 ns (20 degrees C) is in agreement with a rigid binding site that rotates together with the whole, hydrated protein, Using a different technique we have obtained the same results as reported by others (G. Sirokman, T. Wilson and J. W. Hastings, Biochemistry 34, 13074-13081, 1995; V. N. Petushkov, B. G. Gibson and J. Lee, Biochem, Biophys, Res, Commun, 211, 774-779, 1995).