A neutron crystallographic analysis of T6 porcine insulin at 2.1 A resolution.

Neutron diffraction data for T(6) porcine insulin were collected to 2.1 A resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.179. The hydrogen-bonding network of the central core of the hexamer was observed and the charge balance between positively charged Zn ions and their surrounding structure was interpreted by considering the protonation and/or deprotonation states and interactions of HisB10, water and GluB13. The observed double conformation of GluB13 was essential to interpreting the charge balance and could be compared with the structure of a dried crystal of T(6) human insulin at 100 K. Differences in the dynamic behaviour of the water molecules coordinating the upper and lower Zn ions were observed and interpreted. The hydrogen bonds in the insulin molecules, as well as those involving HisB10 and GluB13, are discussed. The hydrogen/deuterium (H/D) exchange ratios of the amide H atoms of T(6) porcine insulin in crystals were obtained and showed that regions highly protected from H/D exchange are concentrated in the centre of a helical region of the B chains. From the viewpoint of soaking time versus H/D-exchange ratios, the amide H atoms can be classified into three categories.

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.