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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 @hen 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 FMN. 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 pkl for the FMN-apoprotein complex at 20°C. The single fluorescence lifetime of YFP-bound FMN is very long (7.6 ns at ZO'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°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, Bwchemishy 34,13074-13081,1995; V. N. Petushkov, B. G. Gibson and J. Lee, Biochem. BWphys. Res. Commun. 21 1, 774-779, 1995).