A Technique for High-Throughput Protein Crystallization in Ionically Cross-Linked Polysaccharide Gel Beads for X-Ray Diffraction Experiments

A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and κ-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K2PtBr6 within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.本研究开发了一种可用于X射线晶体学（X-ray crystallography）中晶体无接触操作的离子交联多糖凝胶微珠内高通量蛋白质结晶技术。该方法旨在降低晶体与晶体固定工具间的物理接触，以及包括冷冻保护（cryoprotection）、重原子衍生化（heavy-atom derivatization）、配体浸泡（ligand soaking）与衍射实验（diffraction experiments）在内的各类操作过程中渗透压冲击（osmotic shock）对晶体造成的机械损伤。本研究以六种测试蛋白开展了海藻酸盐（alginate）与κ-角叉菜聚糖（κ-carrageenan）凝胶微珠内的蛋白质结晶实验，结果证实蛋白质可在凝胶微珠中成功结晶。我们在100K条件下无需取出晶体，即完成了凝胶微珠内溶菌酶（lysozyme）与ID70067蛋白晶体的两套完整衍射数据收集，结果显示此类晶体的镶嵌度（mosaicities）极低。此外，本研究在合成沸石分子筛（synthetic zeolite molecular sieves, MS，一种异质外延成核剂（hetero-epitaxic nucleant））存在的条件下，于海藻酸盐凝胶微珠中完成了葡萄糖异构酶（glucose isomerase）的结晶实验，结果表明MS可抑制该蛋白在微珠内的过量成核（excess nucleation）。为验证重原子衍生化流程，我们成功利用K2PtBr6对海藻酸盐凝胶微珠内的溶菌酶晶体完成了衍生化处理。上述结果表明，凝胶微珠可有效避免此类实验过程中蛋白质晶体遭受的严重损伤。