Consider the thermodynamic cycle given in Figure . To calculate the absolute protein pK a value of a single residue (A), we must consider the free energy of proton transfer from the gas (g) phase into the solution (s) phase and then from the solution into the protein (p) phase. However, for many model compounds, the free energy associated with the proton transfer in solution is known. Using this reference pK a value (pK • a ) allows us to only consider the free energies associated with the rightmost cycle, thus reducing our problem to solving ∆∆G s,p (A H → A -) -the free energy difference between a deprotonation event in the solution phase and in the protein phase -which is related to the protein pK a by