Transition in the temperature-dependence of GFP fluorescence: from proton wires to proton exit.
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.
Leiderman
Pavel
P
Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel-Aviv University, Tel-Aviv, Israel.
Huppert
Dan
D
Agmon
Noam
N
D000818:Animals; D000838:Anions; D003063:Cnidaria; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006860:Hydrogen Bonding; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D015233:Models, Statistical; D011336:Probability; D011522:Protons; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors; D014961:X-Ray Diffraction; D014965:X-Rays
D000838:Anions; D011522:Protons; D049452:Green Fluorescent Proteins