We performed a theoretical study to elucidate the coupling between protonation 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, respectively. In the pH-range from approximately 8 to approximately 11 ionized and neutral sites are present in constant ratio, linked by internal proton transfer. The results indicate that modulation of the internal proton sharing by 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 heterogeneities in the internal hydrogen-bond network lead to distributed driving forces for excited-state proton transfer. A molecular model for the unrelaxed surrounding after deprotonation is discussed in relation to pathways providing fast ground-state recovery or slow stabilization of the anion. The calculated total free energy for excited-state deprotonation ( approximately 19 k(B)T) and ground-state reprotonation ( approximately 2 k(B)T) is in accordance with absorption and emission data (

We present a theoretical study of the four aqueous microscopic dissociation constants relating the relevant protonation forms (cation, neutral, anion, zwitterion) of the chromophore of the green fluorescent protein (GFP) in the ground and excited states. To take the protonation-state-dependent torsional flexibility around the ring-bridging bonds into account, configuration integrals in the torsion space were evaluated to yield the free energy differences. Conformational energies were calculated within a semiempirical quantum chemical scheme using a continuum solvation model. After establishing a linear regression of experimental aqueous pK a 's and calculated enthalpy differences for a series of reference molecules with phenolic hydroxyl or imino nitrogen, the applied method is able to reproduce the titration behavior of bifunctional groups within an average error of 0.8 pK-units. The calculated values for the GFP chromophore agree very well (deviation < 0.2 pK-units) with known ground-state aqueous pK a 's and confirm the equilibrium between neutral and anionic forms (pK a ) 8.3). Freezing torsional flexibility shifts this pK a to 6.6sin accordance with entropic contributions due to the enlarged configurational space of the protonated compoundsand is, therefore, a dominant contribution to the adjustment of pK a 's in the protein. Estimates for the excited-state pK a 's were derived from vertical excitation energies under the assumption that the protolytic equilibria are faster than conformational relaxation of solute and solventscorresponding to a recent experimental model of excited-state proton transfer in GFP. The calculations reveal that increase of both the acidity of the phenolic oxygen and the basicity of the heterocycle nitrogen works in synergy. The neutral form becomes a strong photoacid and transfers a proton with a pK a * ) 0.1; the imino-N becomes a strong photobase capable of accepting a proton with a pK a * ) 8.9. Provided an extended hydrogen-bonded network links the two functions, phototautomerization might be a possible decay pathway.

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