Transition in the temperature dependence of GFP fluorescence:
From proton wires to proton exit
Pavel Leiderman 1, Dan Huppert 1 and Noam Agmon 2*
1 Tel-Aviv Univ.
2 Hebrew University
* To whom correspondence should be addressed. E-mail: agmon{at}fh.huji.ac.il.
Submitted on June 24, 2005
Revised on August 22, 2005
Accepted on 24 October 2005
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Abstract |
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In green fluorescent protein (GFP) 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 Glu222. X-ray data show that the protein contains more extended hydrogen-bonded networks which can support proton migration (``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 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 Thr203, which eventually allows the proton to escape to the exterior solution.
Key Words:
GFP, diffusion, kinetics, proton wires, temperature dependence
