Crystallization of a large single crystal of cubic insulin for neutron protein crystallography

The growth of a large single crystal of cubic porcine insulin for characterization of hydrogen and hydration in cubic insulin crystals by neutron diffraction analysis is reported. Growth in D₂O was investigated based on the phase diagram for cubic insulin to determine appropriate growth conditions, and a large single crystal was then successfully grown by a dialysis method to a size of 4.0 × 4.0 × 1.3 mm³. Neutron diffraction analysis of the cubic insulin crystals was carried out using a single-crystal diffractometer at the JRR-3M reactor of the Japan Atomic Energy Research Institute. In preliminary analysis, N_π appears to be protonated and N_τ deprotonated in His5 in the B-chain, whereas both N_π and N_τ are protonated in His10.