Crystal Growth of a Bilin Reductase PcyA I86D Mutant–Substrate Complex for Neutron Crystallography
| Autor: | Yoshinori Hagiwara, Keiichi Fukuyama, Keisuke Igarashi, Masaki Unno, Katsuhiro Kusaka, Kei Wada, Andreas Ostermann, Naomine Yano, Atsushi Ikeda, Masakazu Sugishima |
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| Rok vydání: | 2018 |
| Předmět: | |
| Zdroj: | Crystal Growth & Design. 18:5174-5181 |
| ISSN: | 1528-7505 1528-7483 |
| DOI: | 10.1021/acs.cgd.8b00607 |
| Popis: | 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 ... |
| Databáze: | OpenAIRE |
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