The determination of protonation states in proteins

The scope of X-ray and neutron protein crystallography to determine the protonation states of functionally important amino acids is investigated at resolutions from 0.94 to 1.50 Å and from 2.2 to 3.75 Å, respectively, using concanavalin A as a test case. Key indicators are then combined to assign levels of confidence of such protonation states for three thrombin X-ray crystal structures at resolutions of 1.26, 1.32 and 1.39 Å. These various studies indicate a widening of the scope of both X-ray and neutron probes in certain circumstances to elucidate the protonation states in proteins.