Abstract
We use state-of-the-art NMR experiments to measure apparent pK(a) values in the native protein environment and employ a cutting-edge combination of enhanced sampling and constant pH molecular dynamics (MD) simulations to rationalize strong pK(a) shifts. The major timothy grass pollen allergen Phl p 6 serves as an ideal model system for both methods due to its high number of titratable residues despite its comparably small size. We present a proton transition analysis as intuitive tool to depict the captured protonation state ensemble in atomistic detail. Combining microscopic structural details from MD simulations and macroscopic ensemble averages from NMR shifts leads to a comprehensive view on pH dependencies of protonation states and tautomers. Overall, we find striking agreement between simulation-based pK(a) predictions and experiment. However, our analyses suggest subtle differences in the underlying molecular origin of the observed pK(a) shifts. From accelerated constant pH MD simulations, we identify immediate proximity of opposite charges, followed by vicinity of equal charges as major driving forces for pK(a) shifts. NMR experiments on the other hand, suggest only a weak relation of pK(a) shifts and close contacts to charged residues, while the strongest influence derives from the dipolar character of α helices. The presented study hence pinpoints opportunities for improvements concerning the theoretical description of protonation state and tautomer probabilities. However, the coherence in the resulting apparent pK(a) values from simulations and experiment affirms cpH-aMD as a reliable tool to study allergen dynamics at varying pH levels.