Proton wires and proton exit in the green fluorescent protein

The Green Fluorescence Protein (GFP) from the jellyfish Aequorea Victoria has attracted much interest as a biological fluorescence marker and an example of excited-state proton transfer (ESPT) in nature. The proton was assumed to migrate irreversibly in the ES to Glu222. In collaboration with Dan Huppert (Tel-Aviv), we have studied the transient fluorescence of the wild-type (wt) protein over a wide range of time and temperature. Unexpectedly, the decay of the acid form of wt-GFP is non-exponential, exhibiting two different power-laws. Below ca. 230 K, we observe a t^-1/2 power-law decay, suggesting one-dimensional diffusion along a pre-formed "proton-wire" within the protein. Indeed, the x-ray structure reveals such a proton-wire, extending beyond Glu-222, on which the proton is apparently trapped at low T. If this interpretation is correct, it provides a first demonstration of proton-wire dynamics in real time.

Above 230 K, the time-resolved kinetics drop below the t^-1/2 asymptotics, exhibiting a t^-3/2 behavior near room-temperature. We show that this phase is not due to three-dimensional diffusion as previously postulated. It stems from an added irreversible sink term to the one-dimensional diffusion equation, depicting irreversible proton exit to solution. The x-ray data reveals that a "threonine switch" (Thr-203) apparently opens above 230 K, establishing a short exit route. The interpretation is corroborated by analysis of the kinetics of the S205V mutant, in which the proton-wire is eliminated, but not the threonine switch. Accordingly, we see in this case slow, exponential proton exit controlled by the conformational change, but no power-laws attributable to diffusion along the proton-wire.

For more information contact Dr. Laren Tolbert (404-894-4093) or Dr. Kyril Solntsev (404-385-4078).