中性子回折による蛋白質単結晶の構造解析

Neutron diffraction experiments for protein

栗原 和男; 岡崎 伸生; 黒木 良太

Kurihara, Kazuo; Okazaki, Nobuo; Kuroki, Ryota

中性子を結晶構造解析に応用すれば、物質中の水素などの軽原子の識別、存在の有無や位置の決定が容易にできる。生命現象の中で蛋白質や水和水中の水素原子の果たす役割は大変重要である。この分野での中性子結晶構造解析では、弱い線源強度を補い効率の良い測定を可能にするための回折装置や試料調製・結晶化技術の開発が成されてきた。日本では、中性子イメージングプレートを検出器として用いた単結晶中性子回折装置BIX-3, BIX-4が建設され、X線データからは予測困難な水素原子位置の決定,水素結合配置の詳細,水和水の持つ配向自由度の様式などの研究に貢献してきた。一方、X線回折との相補的な利用も進み、最近ではX線との同時構造精密化法を用いた創薬標的蛋白質に対する中性子構造解析から、その酵素反応機構の解明や水素がかかわる特殊な立体構造の観察に成功してきている。得られた水素・水和構造の情報は、世界初の生体水素水和水データベースに集約され、水素情報の統計的な様相が解析可能になっている。海外を含め、新たな回折装置,試料調製・結晶化技術や解析手法の開発への取り組みは現在もなお進められており、水素含めた機能解明や研究機会のさらなる拡大を目指している。

Crystallographic analysis using neutron diffraction allows identification and position determination of light atoms like hydrogen. This method has been used for three-dimensional structure determination of organic compounds as well as macromolecules like protein. Hydrogen atoms in proteins, as well as those in solvent molecules, play significant roles in many naturally occurring processes, such as catalytic function and molecular recognition. In the field of neutron crystallography novel diffractometers and techniques for preparation and crystallization of target samples has been developed to complement the low flux of neutron sources to permit higher measurement performance. In Japan, single-crystal diffractometers named BIX-3 and BIX-4 were constructed with Neutron Imaging Plates as a detector. These diffractometers have contributed to the investigation of hydrogen-related molecular structures; for example, determination of hydrogen atom positions which are difficult to predict based on X-ray structure data, precise configuration of hydrogen bonds, and the orientation degree of freedom of hydration water molecules. On the other hand, a complementary application of neutron diffraction with X-ray diffraction has also been developed. Using a joint structure refinement method with X-ray diffraction data, elucidation of an enzymatic reaction mechanism and observation of a particular atomic configuration including hydrogen atoms were successfully achieved in neutron crystallographic studies of drug-discovery-target proteins. The information obtained from these neutron analyses has been consolidated into a database called Hydrogen and Hydration Database for Biomolecules which permits the analysis of key statistical information. In Japan as well as overseas, efforts to acquire higher measurement performance are now in progress to further investigate mechanisms involving hydrogen atoms, and to increase the application of neutron crystallographic studies.