Developing a Method for Locating Ionizable Hydrogen Atom Positions in Protein Crystal Structures.

摘要

Hydrogen is perhaps the most important atom in a protein as it plays a key role in mechanistic enzymology, protein folding, protein engineering, and rational drug design. However, as hydrogen has only one electron, it is rarely observed in an X-ray diffraction experiment. The goal of this work is to develop a means of improving protein models with respect to H-atom placement, with the aim of creating a method of locating hydrogen atoms and determining protonation state for ionizable residues in protein crystal structures that does not exclusively depend on direct visualization of their electron density.;In order to accomplish this, the protein gamma-Chymotrypsin was subjected to study by both neutron and X-ray diffraction. The neutron diffraction studies allowed the direct observation of hydrogen (deuterium) atoms. The X-ray diffraction studies, conducted at ultra-high resolution and encompassing room and liquid nitrogen temperature, hydrogenated and deuterated buffer solutions, and changes in pH conditions, yielded precise information on the distances between ionizable oxygen and nitrogen atoms to atoms to which they are covalently bound as well as the distances between ionizable oxygen and nitrogen atoms to the heavy (non-hydrogen) atoms with which they interact non-covalently.;Neutron diffraction data collected to 2.0 A resolution at pH 5 and pH 7 have successfully identified the positions of hydrogen (deuterium) atoms on ionizable and exchangeable residues in the protein. The ultra-high resolution X-ray diffraction data, collected at a nominal resolution of 1.05 A for most data sets, has yielded the distance information outlined above for aspartate, glutamate, histidine, lysine, serine, threonine, and tyrosine residues. The examination of the covalent distances for aspartate, glutamate, and histidine has shown that these measures are accurate predictors of protonation (deuteration) state. For the remaining four residues, changes in the covalent and non-covalent distances are not accurate predictors of the presence of hydrogen or deuterium. The distances also show that the pK a of carboxylate residues is increased in certain instances at low temperature. This study should promote a re-examination of mechanistic conclusions drawn from liquid nitrogen temperature X-ray diffraction studies.