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 Vi brio fischeri has provided quantitative, thermodynamic information on the FMN-apoYFP equilibrium in aqueous buffer, In diluted aqueous solut ion two fluorescent species could be identified by distinct fluorescen ce lifetimes and rotational correlation times originating from free- a nd protein-bound PMN. Quantitation of the amounts of free and bound FM N 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 ver y long (7.6 ns at 20 degrees C), suggesting a binding environment in w hich maximal emission is provided commensurate with its function as a bioluminescent emitter, The single correlation time of 14.8 ns (20 deg rees C) is in agreement with a rigid binding site that rotates togethe r with the whole, hydrated protein, Using a different technique we hav e obtained the same results as reported by others (G. Sirokman, T. Wil son and J. W. Hastings, Biochemistry 34, 13074-13081, 1995; V. N. Petu shkov, B. G. Gibson and J. Lee, Biochem, Biophys, Res, Commun, 211, 77 4-779, 1995).