PH-Dependent Protonation of the Phl p 6 Pollen Allergen Studied by NMR and cpH-aMD.

Florian Hofer,Valentin Dietrich,Klaus R Liedl,Martin Tollinger,Anna S Kamenik

doi:10.1021/acs.jctc.9b00540

Florian Hofer, Valentin Dietrich + Show 3 more

Open Access

https://doi.org/10.1021/acs.jctc.9b00540

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Abstract

We use state-of-the-art NMR experiments to measure apparent pKa 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 pKa 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 pKa predictions and experiment. However, our analyses suggest subtle differences in the underlying molecular origin of the observed pKa 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 pKa shifts. NMR experiments on the other hand, suggest only a weak relation of pKa 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 pKa values from simulations and experiment affirms cpH-aMD as a reliable tool to study allergen dynamics at varying pH levels.

Highlights

The pH level is well known to be a critical environmental determinant of protein function and stability.[1−3] Small changes in solution pH can be sufficient to completely destabilize a protein or change its activity profile.[4,5] Proteins react to pH changes via protonation or deprotonation of titratable residues, thereby changing their charge distribution
Combining cpH-aMD simulations and NMR experiments, we present a strategy for detailed studies on the titration behavior of proteins
For 11 out of the 18 titrated residues, the difference between experiment and simulation is less than 1 pKa unit, an error margin typically reported in the literature

Summary

Introduction

The pH level is well known to be a critical environmental determinant of protein function and stability.[1−3] Small changes in solution pH can be sufficient to completely destabilize a protein or change its activity profile.[4,5] Proteins react to pH changes via protonation or deprotonation of titratable residues, thereby changing their charge distribution. The nature and strength of this effect depends on the number of affected titratable residues, on their structural environment, and on possible compensation of introduced charges. The acidity of a titratable group is represented by its pKa value and directly dependent on its electrostatic surroundings.[6,7] while for isolated amino acids in solution these pKa values are measurable, they can be drastically perturbed and challenging to measure within the context of a protein.[8] Yet, an accurate representation of protonation states and tautomers is paramount for reliable experimental and especially computational studies of protein structures.[9]

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