The Effect Of Different Gravity Levels Simulated By Strong Magnetic Field On Protein Crystallization

Protein structure and function have great scientific significance on exploring the mysteries of life, developing of new drugs. The structure decides the function of proteins. X-ray diffraction(XRD) has determined 88% protein structures of Protein Data Bank(PDB) as one of the key technology. It has been the bottleneck problem of XRD to obtain high quality crystals. It is necessary to explore various methods to improve the quality of protein crystals.The dissertation aims to explore new methods and devices to improve the quality of protein crystal in strong magnetic field to provide a new solution for the bottleneck problem. The researches were conducted as follows:1. The effect of different gravity levels simulated by strong magnetic field on the protein crystal quality. The different gravity levels such as 0 g?1 g?2 g levels were simulated by strong magnetic field. The normal gravity level outside the magnet was as the control. For the first time, we studied systematically effects of different gravity levels simulated by large gradient strong magnetic field on the protein crystal quality by analyzing the diffraction quality of six protein crystals grown in the four crystallization conditions. The highest resolution, mosacity and R factor were choosed as the factor of crystal quality comparison. It was indicated that the morphology and crystal quality of were improved under simulated 0 g, 1 g and 2 g levels compared with the control. Among them, simulated 0 g exhibited the best improvement, 1 g in the second one and 2 g in the third one. It was demonstrated that simulated 0 g level have ability to improve crystal quality, which can provide new solution for the bottleneck problem of protein structure determination.2. The device and method of high-throughput protein crystallization based on the capillary array. Normal commercial crystallization plates cannot been applied to special enviroment such as strong magnetic fields. As a result, cylindrical high-throughput protein crystallization device was developed to solve the problem. The capillary array can be applied to small space to realize high-throughput protein crystallization and screening, which took advantage of its characteristic of small size. The device can not only be applied to normal environment but also special physicalenvironment such as magnetic field, electrical field and space to realize high-throughput protein crystallization and screening. For the first time, predrying liquid-solid crystallization method was put forward based on the capillary. The innovation reflected on predrying protein solution in the capillary and then mixed with screening reagents. The method was different with all other crystallization methods, which can provide a new crystallization environment to obtain new crystallization conditions.3. The application of high-throughput crystallization device based on capillary array in strong magnetic field. Lysozyme, proteinase K and glucose isomerase were used as target proteins. For the first time, the high-throughput crystallization and screening were realized successfully in the strong magnet using high-throughput crystallization device based on capillary array. The in-situ diffraction of crystals was also realized successfully.In summary, the major results obtained in this study are as follows:1. For the first time, the effect of different gravity levels simulated by strong magnetic field on protein crystallization was systematically studied. It was indicated that the crystal quality of were improved under simulated 0 g, 1 g and 2 g levels compared with the control. Among them, simulated 0 g exhibited the best improvement, 1 g in the second and 2 g in the third one. It was demonstrated that simulated 0 g level have ability to improve crystal quality, which can provide new solution for the bottleneck problem of protein structure determination.2. The cylindrical high-throughput protein crystallization device based on capillary array was developed. The device can not only be applied to normal environment but also special physical environment such as magnetic field, electrical field and space to realize high-throughput protein crystallization and screening.3. For the first time, predrying liquid-solid crystallization method was put forward based on the capillary. The method was different with all other crystallization methods, which can provide a new crystallization environment to obtain new crystallization conditions.4. For the first time, the high-throughput crystallization and screening were realized successfully in the strong magnet using high-throughput crystallization device based on capillary array. The in-situ diffraction of crystals was also realized successfully.