Spatially Precise, Soft Microseeding of Single Protein Crystals by Femtosecond Laser Ablation

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• 作者：Hiroshi Y. Yoshikawa ; Yoichiroh Hosokawa ; Ryota Murai ; Gen Sazaki ; Tomoya Kitatani ; Hiroaki Adachi ; Tsuyoshi Inoue ; Hiroyoshi Matsumura ; Kazufumi Takano ; Satoshi Murakami ; Seiichiro Nakabayashi ; Yusuke Mori ; Hiroshi Masuhara
• 刊名：Crystal Growth & Design
• 出版年：2012
• 出版时间：September 5, 2012
• 年：2012
• 卷：12
• 期：9
• 页码：4334-4339
• 全文大小：356K
• 年卷期：v.12,no.9(September 5, 2012)
• ISSN：1528-7505

文摘

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 渭m²) 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.