卵白リゾチームのガラス基板上への不均一核形成と単結晶育成

タンパク質の立体構造を解析する方法は、現在ではX線結晶構造解析が最も優れている。しかしながら、X線結晶解析における最も困難なプロセスは、良質なタンパク質の単結晶育成である。タンパク質の結晶成長機構にはまだ不明な点も多く、基礎的な立場からタンパク質結晶成長の要素解析が必要である。そこで本研究では卵白リゾチームを用い、水溶液中に挿入したガラス基板上における不均一核形成現象を観察すると同時に、ガラス基板を用いた大きな単結晶育成を試みた。その結果、斜方晶多形の単結晶が成長し、析出剤濃度が高く、pH8.0近傍の条件で、不均一核形成が促進されることが判明した。さらに、ガラス基板上において、斜方晶結晶の凝集はまれにしか起こらないうえに、基板を傾斜させた方がより不均一核形成を制御しやすいことが明かになった。このような不均一核形成現象を利用して育成した大きな単結晶を用い、結晶のキャラクタリゼーションなどのタンパク質結晶化のメカニズムを解明することが可能となった。

X-ray structural analyses for molecular structures of proteins have been most convincing and informative. In the course of the X-ray analysis, growth of protein crystals of high quality is the most difficult process and the mechanisms of protein crystal growth have been less known in comparison to small molecule crystals. Hence, fundamental studies are highly needed to fully understand the protein crystallization processes. In this study, we tried to crystallize large-size single crystals on glass plates which were inserted in a growth solution, using hen egg-white lysozyme as model of protein. Heterogeneous nucleation process played an important role in this crystallization technique. The following results were obtained : (1) Orthorhombic form single crystals were grown under the present growth conditions (growth temperature of 35℃, and pH8.0), (2) heterogeneous nucleation was promoted at higher concentration of precipitant (NaCl), (3) crystals were grown on the glass plates, (4) it was easier to grow large crystals by inclining the glass plates in the solution, (5) single crystals having the maximum dimensions of 10 mm in length and 3 mm in width and 2 mm thickness could be crystallized. Using the large-size single crystals obtained with this method, one can characterize the protein crystals such as crystal perfection, surface microtopography, and crystal growth process etc.