Electrostatics play an important role in many aspects of protein chemistry. However, the accurate determination of side chain proton affinity in proteins by experiment and theory remains challenging. In recent years the field of nuclear magnetic resonance spectroscopy has advanced the way that protonation states are measured, allowing researchers to examine electrostatic interactions at an unprecedented level of detail and accuracy. Experiments are now in place that follow pH-dependent 13C and 15N chemical shifts as spatially close as possible to the sites of protonation, allowing all titratable amino acid side chains to be probed sequence specifically. The strong and telling response of carefully selected reporter nuclei allows individual titration events to be monitored. At the same time, improved frameworks allow researchers to model multiple coupled protonation equilibria and to identify the underlying pH-dependent contributions to the chemical shifts.


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