Crystal Growth of a Bilin Reductase PcyA I86D Mutant–Substrate Complex for Neutron Crystallography,Crystal Growth & Design

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Crystal Growth of a Bilin Reductase PcyA I86D Mutant–Substrate Complex for Neutron Crystallography
Crystal Growth & Design ( IF 3.2 ) Pub Date : 2018-07-11 00:00:00 , DOI: 10.1021/acs.cgd.8b00607
Keisuke Igarashi _{1,

2} , Yoshinori Hagiwara ₃ , Masakazu Sugishima ₄ , Kei Wada ₅ , Keiichi Fukuyama ₆ , Atsushi Ikeda ₁ , Naomine Yano ₂ , Katsuhiro Kusaka ₂ , Andreas Ostermann ₇ , Masaki Unno _{1,

2}

Affiliation

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.

中文翻译：

中子晶体学的Bilin还原酶PcyA I86D突变体-基质复合物的晶体生长。

蛋白质的中子晶体学比X射线晶体学需要更大的晶体。获得酶-底物复合物的大晶体特别困难，因为晶体生长需要严格控制化学计量。本文报道了一种获得晶体的程序，该晶体足够大以对与其底物biliverdin（一种开放的四吡咯颜料）复合的bilin还原酶PcyA I86D突变体进行中子结构分析。通过观察吸收光谱的变化估算酶/底物的比率，并将其与理论值进行比较；在实验中需要1.2倍的胆绿素含量。PcyA I86D突变体/胆小素的这种适当的化学计量比可重现，可得到高质量的晶体。通过分析使用三种不同缓冲液获得的晶体的室温X射线数据的Wilson图，选择了坐滴蒸汽扩散法中用于储层溶液的缓冲液。PcyA I86D突变体/胆红素复合物与结晶溶液的混合比最优化为9：1。这些结果表明，优化酶/底物比例以及结晶溶液可以是大晶体生长的有效策略。

更新日期：2018-07-11

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