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Transition in the Temperature-Dependence of GFP Fluorescence: From Proton Wires to Proton Exit

Pavel Leiderman ^*, Dan Huppert ^* and Noam Agmon 

^* Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel-Aviv, Israel; and Department of Physical Chemistry and the Fritz Haber Research Center, The Hebrew University, Jerusalem, Israel

Correspondence: Address reprint requests to Dr. Noam Agmon, Tel.: 972-2-658-5687; Fax: 972-2-651-3742; E-mail: agmon{at}fh.huji.ac.il.

In green fluorescent protein, photo-excitation leads to excited-state proton transfer from its chromophore, leaving behind a strongly fluorescing anion, while the proton is commonly thought to migrate internally to Glu-222. X-ray data show that the protein contains more extended hydrogen-bonded networks that can support proton migration (i.e., proton wires). Here we study the temperature-dependence of the transient fluorescence from both the acid and anionic forms up to 15 ns. At low temperatures, we find that the (lifetime-corrected) fluorescence of the acidic form decays asymptotically as t^–1/2, following quantitatively the solution of a one-dimensional diffusion equation for reversible geminate recombination with quenching. This indicates proton migration along the internal proton wires. A small degree of geminate proton quenching is attributed to the formation of the zwitterion by proton migration on a side-branch of the proton wire. Above 230 K, the fluorescence kinetics undergo a transition, exhibiting an asymptotic t^–3/2 decay, and the quenching effect disappears. We interpret these findings as evidence for a conformational change enabling the rotation of Thr-203, which eventually allows the proton to escape to the exterior solution.

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