The green fluorescent protein (GFP) is the only naturally occurring protein in which excited-state proton-transfer has been identified. Upon excitation, a proton is ejected from the phenolic moiety of its chromophore, travelling through the 'privileged water molecule' (PWM) to Glu222 in about 10 ps. However, time-resolved fluorescence from the acidic form exhibits a t-a power-law decay (times an exponential radiative decay) extending well into the ns regime. With increasing temperature, a switches from 1/2 (below 230 K) to 3/2 (above it). This has been interpreted as pseudo one-dimensional proton hopping along an internal 'proton wire', with an activated process that opens a 'doorway' for proton escape to solution at the higher temperatures [1].To identify such putative pathways, we have developed a computer code that maps all the 'proton wires' within a given protein structure, applying it to a X-ray GFP structure of 0.9 � resolution [2]. A proton wire indeed continues from Glu222 along the axis of the GFP 'barrel', connecting to a negatively charged surface patch ('proton collecting antenna'?). This might explain the t-1/2 behavior. However, a direct escape pathway from the chromophore to solution (explaining the switchover to t-3/2) is not easily identified in the X-ray structure.We conducted molecular dynamics simulations showing that the PWM can exit to solution on the 100 ps timescale. This occurs by beta-sheet fluctuations, creating an opening through which water molecules can leave and enter the protein. The water-exchange kinetics follows a stretched-exponential time behavior, whereas its mean squared displacement along the escape pathway shows a transition from subdiffusion to superdiffusion and then to normal diffusion (in the bulk). We ascribe this to a flip-flop mechanism by which the water traverses the protein.[1] Agmon, J. Phys. Chem. B 2007, 111 , 7870.[2] Shinobu et al., JACS 2010, 132  , 11093.

Proton migration and privileged water exchange in the green fluorescent protein

248th National Meeting (2014)

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