A neutron crystallographic analysis of phosphate-free ribonuclease A at 1.7 A resolution.

A neutron crystallographic analysis of phosphate-free bovine pancreatic RNase A has been carried out at 1.7 A resolution using the BIX-4 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Agency. The high-resolution structural model allowed us to determine that His12 acts mainly as a general base in the catalytic process of RNase A. Numerous other distinctive structural features such as the hydrogen positions of methyl groups, hydroxyl groups, prolines, asparagines and glutamines were also determined at 1.7 A resolution. The protonation and deprotonation states of all of the charged amino-acid residues allowed us to provide a definitive description of the hydrogen-bonding network around the active site and the H atoms of the key His48 residue. Differences in hydrogen-bond strengths for the alpha-helices and beta-sheets were inferred from determination of the hydrogen-bond lengths and the H/D-exchange ratios of the backbone amide H atoms. The correlation between the B factors and hydrogen-bond lengths of the hydration water molecules was also determined.

Crystallization and evaluation of hen egg-white lysozyme crystals for protein pH titration in the crystalline state.

To observe the ionized status of the amino acid residues in proteins at different pH (protein pH titration in the crystalline state) by neutron diffraction, hen egg-white lysozyme was crystallized over a wide pH range (2.5-8.0). Crystallization phase diagrams at pH 2.5, 6.0 and 7.5 were determined. At pH < 4.5 the border between the metastable region and the nucleation region shifted to the left (lower precipitant concentration) in the phase diagram, and at pH > 4.5 the border shifted to the right (higher precipitant concentration). The qualities of these crystals were characterized using the Wilson plot method. The qualities of all crystals at different pH were more or less equivalent (B-factor values within 25-40). It is expected that neutron diffraction analysis of these crystals of different pH provides equivalent data in quality for discussions of protein pH titration in the crystalline state of hen egg-white lysozyme.