Investigation of light-induced nucleation mechanism of proteins

后基因组研究的一个主题是确定蛋白质的3D结构。这项研究的主要技术是X射线结晶学。自从同步辐射被利用以来，X射线结晶学在分析晶体方面取得了很大的进展。然而，制备高质量单晶的技术仍在日益进步。我们演示了光化学诱导成核的鸡蛋清溶菌酶成核在氙灯照射几十秒。1)改进了实验方法，重复性好；2)探索了更适合于光诱导成核的结晶配方；3)除鸡蛋溶菌酶外，其他蛋白质的光诱导成核。[结果]1)改进了实验方法。蛋白质结晶实验一般采用气相扩散法，如悬滴法或静滴法。…更多的是，我们首先尝试将光导照射到玻璃板上的过饱和溶液中。虽然观察到了成核增强，但重复性较差。这是由于氦液滴在气液界面蒸发而导致的不均匀成核所致。为了避免这种不确定性，我们改进了实验程序，如图所示。结果，我们得到了很好的重现性，即在没有光照的情况下，溶液中不发生成核；另一方面，在辐照的溶液中发生了成核。2)研究了光诱导成核的结晶配方。我们研究了适合于光诱导成核的结晶配方。在相同的过饱和度下，聚乙二醇4000(PEG4000)比不含PEG4000的聚乙二醇4000更有效地促进光诱导成核。作为蛋鸡溶菌酶以外的另一种蛋白质，进行了索马汀的结晶。测定了其溶解度曲线。即使在不饱和溶液中，成核作用也是暂时的。[结论]改进了实验方法。对几种商业上可获得的蛋白质进行了光诱导成核实验。我们还计划开发工业蛋白质结晶系统。较少

One subject for post genome research is to determine protein 3D structure. The main technique for this research is X-ray crystallography. X-ray crystallography has experienced great progress in analyzing a crystal since synchrotron radiation has been utilized. Whereas, the technique for the preparation of high quality single crystal is still progress day by day. We demonstrate photochemically-induced nucleation of hen egg-white lysozyme nucleation by Xe-lamp irradiation for few tens seconds. We have demonstrated nucleation enhancement mechanism.^<1)> We report here, 1) improvement of the experimental procedure with good reproducibility, 2) investigation of crystallization recipes that are more suitable for light-induced nucleation, and 3) light-induced nucleation of proteins except for hen egg lysozyme.【Results】1)Improvement of the experimental procedure. Protein crystallization experiments have been generally carried out by vapor diffusion such as hanging drop or sitting drop method. … More We, first, tried to irradiate light directory to supersaturated solution on a glass plate. Though, nucleation enhancement was observed, reproducibility was poor. This was result from undesired nucleation by evaporation from vapor-solution interface of he droplets, which induce inhomogeneous nucleation. To avoid such uncertainties, we improved experimental procedures illustrated in Figure. As a result, we obtained good reproducibility that nucleation did not take place in the solution without light irradiation ; on the other hand nucleation took place with irradiated solution.2) Investigation of crystallization recipes for light-induced nucleation. We investigated crystallization recipes suitable for light-induced nucleation. Polyethylene glycol 4000 (PEG4000) enhanced more effectively light-induced nucleation than without containing PEG4000 at the same supersaturation.3)Crystallization of thaumatin. As another protein except for hen-egg lysozyme, crystallization of thaumatin was carried out. Solubility curve was determined. Nucleation enhancement took place temporary even in undersaturated solution. This suggests that molecular interaction between thaumatin changes attractively by the irradiation.【Conclusion】Experimental procedure was improved. Light-induced nucleation experiments are carried out for several commercially obtainable proteins. We are also planning to develop industrial protein crystallization system. Less