Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: Protonation and conformational substates from electrostatic calculations

We performed a theoretical study to elucidate the coupling between protonat ion states and orientation of protein dipoles and buried water molecules in green fluorescent protein, a versatile biosensor for protein targeting. It is shown that the ionization equilibria of the wild-type green fluorescent protein-fluorophore and the internal proton-binding site E222 are mutually interdependent. Two acid-base transitions of the fluorophore occur in the presence of neutral (physiologic pH) and ionized (pH > 12) E222, respective ly. In the pH-range from approximate to 8 to approximate to 11 ionized and neutral sites are present in constant ratio, linked by internal proton tran sfer. The results indicate that modulation of the internal proton sharing b y structural fluctuations or chemical variations of aligning residues T203 and S65 cause drastic changes of the neutral/anionic ratio-despite similar physiologic fluorophore pK(a) s. Moreover, we find that dipolar heterogenei ties in the internal hydrogen-bond network lead to distributed driving forc es for excited-state proton transfer. A molecular model for the unrelaxed s urrounding after deprotonation is discussed in relation to pathways providi ng fast ground-state recovery or slow stabilization of the anion. The calcu lated total free energy for excited-state deprotonation (approximate to 19 k(B)T) and ground-state reprotonation (approximate to 2 k(B)T) is in accord ance with absorption and emission data (less than or equal to 5000 cm(-1) o r 24 k(B)T).