Amino acids with ionizable side chains make up approximately 30% of the residues found in proteins and play a key role in maintaining protein stability , modulating solubility , mediating protein-protein interactions , and facilitating cell signaling . These amino acids, namely aspartate, glutamate, arginine, lysine, cysteine, tyrosine, and histidine, are functionally dependent on their protonation states, which vary depending on their local environments. The measure of this dependence is known as the pK a , which relates the pH of the solution to the protonation state of a residue, via the Henderson-Hasselbalch equation, that is, pK a = pH + log [HA]/[A -]. Given its degree of solvent exposure, Coulombic interactions, and hydrogen bonding, the pK a of an amino acid residue may be raised or lowered relative to its reference pK • adetermined using a blocked peptide (e.g., ACE-AXA-NH 2 ) in solution -resulting in a lower or higher likelihood of protonation at a given pH. For acidic groups, the pK a values tend to be elevated relative to their reference , while for basic groups, the pK a values tend to be lowered relative to their reference . These shifts away from the reference value can be on the order of ±5 pK units, and in many proteins, key ionizable residues are situated in such a way that a perturbation of their pK a allows them to perform unique and specific functions . The existence of such functional motifs relies on the alterable stability of the covalent bond between the hydrogen and its heavy atom (e.g., O-H and N-H). The tendency of a side chain containing these groups to (de)protonate in a given microenvironment is quantified by the pK a .