Spatially precise, soft microseeding of single protein crystalsby femtosecond laser ablation
Spatially precise, soft microseeding of single protein crystalsby femtosecond laser ablation
复制标题
通过飞秒激光烧蚀对单个蛋白质晶体进行空间精确、软微播种
DOI：
10.1021/cg300018t
复制
发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Hiroshi Yoshikawa
中科院分区：
文献类型：
--
作者：
S.Nakayama;H.Y.Yoshikawa;R.Murai;M.Kurata;M.Maruyama;S.Sugiyama;Y.Aoki;Y.Takahashi;M.Yoshimura;S.Nakabayashi;H.Adachi;H.Matsumura;T.Inoue;K.Takano;S.Murakami;and Y.Mori;Hiroshi Yoshikawa
我们开发了一种空间精确的、软的微种方法来生产适合于X射线结晶学研究的单蛋白质晶体。我们使用聚焦的飞秒激光脉冲，通过多光子吸收过程，从小区域(∼1μm2)的晶体中产生种子晶体。以这种方式生产的蛋清溶菌酶种子晶体，在结晶度没有任何恶化的情况下成长为单晶。我们还验证了使用多晶技术来获得膜蛋白-阿克利黄素抗性蛋白B的技术，对于这种蛋白来说，单晶是很难获得的。此外，我们还发现，通过改变晶化开始和激光烧蚀之间的时间间隔，可以控制由种子制备的四方溶菌酶晶体的形状。我们还试图理解飞秒激光诱导微种子产生的机理。我们用原子力显微镜和激光共聚焦显微镜结合差示干涉显微镜显示了溶菌酶晶体的烧蚀表面。这项研究的结果清楚地表明，飞秒激光烧蚀蛋白质晶体是基于光机械过程，该过程射出晶体碎片，热损伤很小。飞秒激光烧蚀确实非常有希望从不适合X射线衍射研究的多晶体或破裂晶体中产生高质量的蛋白质种子晶体。
We developed a spatially precise, soft microseeding method for the production of single protein crystals that are suitable for X-ray crystallographic studies. We used focused femtosecond laser pulses to produce, via multiphoton absorption processes, seed crystals from small regions (∼1 μm2) of crystals. Hen egg-white lysozyme seed crystals, produced in this manner, grew to be single crystals without any deterioration in their crystallinity. We also validated the technique using polycrystals for the membrane protein, acriflavine resistance protein B, for which single crystals are very difficult to obtain. In addition, we found that the shape of a tetragonal lysozyme crystal prepared from the seed could be controlled by altering the time interval between the initiation of crystallization and laser ablation. We also tried to comprehend the mechanism of femtosecond laser-induced microseeding. We visualized the ablated surfaces of the lysozyme crystals by atomic force microscopy and by laser confocal microscopy combined with differential interference microscopy. The results obtained in this study clearly demonstrate that femtosecond laser ablation of protein crystals is based on a photomechanical process, which ejects crystal fragments with little thermal damage. Femtosecond laser ablation is indeed very promising to produce high quality protein seed crystals from polycrystals or cracked crystals that are not suitable for X-ray diffraction studies.