Investigation of light-induced nucleation mechanism of proteins

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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