Ultrafast Electron Transfer in the Complex between Fluorescein and a Cognate Engineered Lipocalin Protein, a So-Called Anticalin

Abstract

Anticalins are a novel class of engineered ligand-binding proteins with tailored specificities derived from the lipocalin scaffold. The anticalin FluA complexes fluorescein as ligand with high affinity, and it effects almost complete quenching of its steady-state fluorescence. To study the underlying mechanism, we have applied femtosecond absorption spectroscopy, which revealed excited-state electron transfer within the FluA‚Fl complex to be responsible for the strong fluorescence quenching. On the basis of a comparison of redox potentials, either tryptophan or tyrosine may serve as electron donor to the bound fluorescein group in its excited singlet state, thus forming the fluorescein trianion radical within 400 fs. The almost monoexponential rate points to a single, well-defined binding site, and its temperature independence suggests an (almost) activationless process. Applying conventional electron transfer theory to the ultrafast forward and slower back-rates, the resulting electronic interaction is rather large, with ∼140 cm -1 for tyrosine, which would be consistent with a coplanar arrangement of both aromatic moieties within van der Waals distance. The weak residual steady-state fluorescence originates from a small (∼10%) component with a time constant in the 40-60 ps range. These results demonstrate the power of timeresolved absorption spectroscopy as a diagnostic tool for the elucidation of a fluorescence quenching mechanism and the temporal profiles of the processes involved. The high structural and dynamic definition of the complexation site suggests the anticalin FluA to be a promising model in order to tailor and probe electronic interactions and energetics in proteins. † M.E.M.-B. gratefully acknowledges financial support by the Deutsche Forschungsgemeinschaft (SFB 533) and the Volkswagenstiftung. A.S. thanks the Fonds der Chemischen Industrie for financial support.

Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers

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Investigating Tryptophan Quenching of Fluorescein Fluorescence under Protolytic Equilibrium

“…The Coulombic interaction experienced by the reactants and products as they are brought together in the encounter pair are included in equation 10 as being Götz et al 45 applied femtosecond absorption spectroscopy to show that fluorescein is electron photoreduced by either tryptophan or tyrosine after binding to Anticalin, a Lipocalin protein, where the fluorescein trianion radical is formed very quickly in about 400 fs. The G 0 value used by Götz et al is in the same range as found in our work.…”

Temperature and pressure dependence of protein stability: The engineered fluorescein-binding lipocalin FluA shows an elliptic phase diagram

“…Interestingly, the characteristic stationary fluorescence of fluorescein becomes al-most completely quenched on complex formation with FluA (26). This phenomenon has been attributed to an extremely fast and efficient electron transfer between the excited fluorescein dianion and a tryptophan side chain that is tightly packed against its xanthenolone moiety in the ligand pocket, followed by a radiationless backtransfer (29).…”

“…In the native state emission of the complexed fluorescein is completely quenched, whereas in the denatured state fluorescein regains its high quantum yield (29). Consequently, measurement of the relative fluorescence intensity as a function of pressure at a defined temperature yields the equilibrium constant for denaturation as a function of pressure or vice versa and, hence, the boundary of the phase diagram.…”