Solution pK_a Values of the Green Fluorescent Protein Chromophore from Hybrid Quantum-Classical Calculations

Abstract

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.

An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP

“…35 Upon electronic excitation, the pK a of the chromophore is reduced. 39,40 In the case of wtGFP the effect is to make the anionic I* form more stable. However, the proton transfer proceeds from A* to I* through a barrier, leading to the observed picosecond lifetime of the A* and its sensitivity to isotopic exchange.…”

Cis–trans photoisomerization of the chromophore in the green fluorescent protein variant E2GFP: A molecular dynamics study

“…For the diabatic potential in the CTP simulations we chose a barrier of 4 kcal/mol around /, from the excited-state energy profile of / torsion reported in [17]. Another semiempirical calculation yields a larger value for the maximum at / = 90°(around 10 kcal/ mol [31]). We made some CTP trial runs using this value, and observed only small deviations from our results with the lower value.…”

Theoretical calculations of the relative pKa values of some selected aromatic arsonic acids in water using density functional theory

“…21,[24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] Recently quantum chemical calculations, using thermodynamical parameters such as Gibbs free energy, have reported good correlations between calculated and experimental acidities of a wide range of organic and inorganic acids. 18,[42][43][44][45][46][47][48][49][50] For example, gas phase and solution phase acidities of some organic compounds investigated by Namazian et al and Kheirjou et al reported good correlation between ab initio calculations of pKa values with the experimental data. 19,21,[51][52] However, to the best of our knowledge, there is no data on pKa calculations of arsonic acids by quantum chemical methods, and experimentally determined pKa values are limited to a small group of arsonic acid derivatives.…”