Crystal Growth of a Bilin Reductase PcyA I86D Mutant-Substrate Complex for Neutron Crystallography

Neutron crystallography of proteins requires much larger crystals than those for X-ray crystallography. Obtaining large crystals of an enzyme-substrate complex is especially difficult because strict control of the stoichiometry is needed for crystal growth. Herein is reported a procedure for obtaining crystals large enough for neutron structural analysis of a bilin reductase PcyA I86D mutant complexed with its substrate, biliverdin, an open tetrapyrrol pigment. The enzyme/substrate ratio was estimated by observing changes in absorption spectra and compared to the theoretical value; 1.2 times the amount of biliverdin was required in the experiment. This proper stoichiometric ratio of PcyA I86D mutant/biliverdin reproducibly gave high-quality crystals. The buffer for the reservoir solution in the sitting drop vapor diffusion method was chosen by analysis of Wilson plots of room temperature X-ray data of the crystals obtained using three different buffers. The mixing ratio of the PcyA I86D mutant/biliverdin complex and the crystallization solution was optimized at 9:1. These results demonstrate that optimization of the enzyme/substrate ratio as well as the crystallization solution can be an effective strategy for large crystal growth.