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Automated high throughput protein crystallization screening at nanoliter scale and protein structural study on lactate dehydrogenase

机译：纳升规模的自动化高通量蛋白质结晶筛选和乳酸脱氢酶的蛋白质结构研究

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The purposes of our research were: (1) To develop an economical, easy to use, automated, high throughput system for large scale protein crystallization screening. (2) To develop a new protein crystallization method with high screening efficiency, low protein consumption and complete compatibility with high throughput screening system. (3) To determine the structure of lactate dehydrogenase complexed with NADH by x-ray protein crystallography to study its inherent structural properties;Firstly, we demonstrated large scale protein crystallization screening can be performed in a high throughput manner with low cost, easy operation. The overall system integrates liquid dispensing, crystallization and detection and serves as a whole solution to protein crystallization screening. The system can dispense protein and multiple different precipitants in nanoliter scale and in parallel. A new detection scheme, native fluorescence, has been developed to form a two-detector system with a visible light detector for detecting protein crystallization screening results. This detection scheme can eliminate common false positives by distinguishing protein crystals from inorganic crystals in a high throughput and non-destructive manner. The entire system from liquid dispensing, crystallization to crystal detection is essentially parallel, high throughput and compatible with automation. The system was successfully demonstrated by lysozyme crystallization screening;Secondly, we developed a new crystallization method with high screening efficiency, low protein consumption and compatibility with automation and high throughput. A gas permeable membrane is employed to achieve the gentle evaporation required by protein crystallization. Protein consumption is significantly reduced to nanoliter scale for each condition and thus permits exploring more conditions in a phase diagram for given amount of protein. The protein crystals gotten by this method were experimentally proven to possess high x-ray diffraction qualities;Finally, we crystallized human lactate dehydrogenase 1 (H4) complexed with NADH and determined its structure by x-ray crystallography;The structure of LDH/NADH displays open, closed conformation or two-conformation on the active site while the subunits in LDH/NADH/inhibitor are all in close conformation. Difference in subunit conformation was observed among the structures independently solved from multiple individual LDH/NADH crystals;Structural differences observed among crystals suggest the existence of multiple conformers in solution.
机译：我们的研究目的是：（1）开发一种经济，易于使用，自动化，高通量的大规模蛋白质结晶筛选系统。 （2）开发一种筛选效率高，蛋白质消耗少，与高通量筛选系统完全兼容的新型蛋白质结晶方法。 （3）通过X射线蛋白质晶体学确定乳酸脱氢酶与NADH复合的结构，以研究其固有的结构特性。首先，我们证明了可以高产量，低成本，易于操作进行大规模蛋白质结晶筛选。整个系统集成了液体分配，结晶和检测功能，是蛋白质结晶筛选的整体解决方案。该系统可以纳升级和并行分配蛋白质和多种不同的沉淀剂。已经开发出一种新的检测方案，即天然荧光，以形成带有可见光检测器的两个检测器系统，用于检测蛋白质结晶筛选结果。该检测方案可以通过以高通量和非破坏性的方式将蛋白质晶体与无机晶体区分开来消除常见的假阳性。从液体分配，结晶到晶体检测的整个系统基本上是平行的，高通量并与自动化兼容。通过溶菌酶结晶筛选成功地证明了该系统；其次，我们开发了一种新型的筛选方法，该方法具有较高的筛选效率，较低的蛋白质消耗量以及与自动化和高通量的相容性。使用透气膜来实现蛋白质结晶所需的温和蒸发。每种条件下的蛋白质消耗量都显着降低至纳升规模，因此可以在相图中探索更多条件下的蛋白质量。通过实验证明了通过该方法获得的蛋白质晶体具有较高的X射线衍射质量；最后，我们将与NADH复合的人乳酸脱氢酶1（H4）结晶，并通过X射线晶体学确定其结构； LDH / NADH显示器的结构在LDH / NADH /抑制剂中的亚基都处于紧密构象时，活性位点上的开放，闭合构象或二构象。从多个单独的LDH / NADH晶体独立解析的结构之间观察到亚基构象差异；在晶体之间观察到的结构差异表明溶液中存在多个构象异构体。

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    Li, Fenglei;

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  • 年度 2006
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  • 正文语种 en
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