When a protein folds, the titratable amino acids in the protein are transferred from a solution-like environment to an environment determined by the 3-dimensional structure of the protein. For example, in an unfolded protein, an aspartic acid typically is in an environment which exposes the titratable side chain to water. When the protein folds, the aspartic acid could find itself buried deep in the protein interior with no exposure to solvent.
Furthermore, in the folded protein, the aspartic acid will be closer to other titratable groups in the protein and will also interact with permanent charges (e.g. ions) and dipoles in the protein. All of these effects alter the pK value of the amino acid side chain, and pK calculation methods generally calculate the effect of the protein environment on the model pK value of an amino acid side chain.
Typically, the effects of the protein environment on the amino acid pK value are divided into pH-independent effects and pH-dependent effects. The pH-independent effects (desolvation, interactions with permanent charges and dipoles) are added to the model pK value to give the intrinsic pK value. The pH-dependent effects cannot be added in the same straightforward way and have to be accounted for using Boltzmann summation, Tanford–Roxby iterations or other methods.
The interplay of the intrinsic pK values of a system with the electrostatic interaction energies between titratable groups can produce quite spectacular effects such as non-Henderson–Hasselbalch titration curves and even back-titration effects.
The image on the right shows a theoretical system consisting of three acidic residues. One group is displaying a back-titration event (blue group).