The acquisition of protein crystals is a crucial step in the analysis of three-dimensional structure of proteins,the production of medical drugs and the construction of self-assembled nanostructures.For example,when we use X-ray diffraction tech-nology to determine the three-dimensional structure of protein,first of all,we need to obtain the high quality of protein crystals.Pro-tein crystallization has two steps,nucleation and growth.The progress from the undersaturation zone through metastable zone to nu-cleation zone is called nucleation.When the protein crystal nucleus passes through the nucleation zone to the metastable zone,the crystal nucleus grows and ripening occurs.In the process of protein crystallization,formation of protein crystal nucleus is fundamen-tal.The whole crystallization system has the same probability to nucleate in homogeneous nucleation process.In this process,the protein starts to nucleate in the nucleation zone only when the degree of supersaturation of the protein solution crystallization system could overcome the nucleation barrier.Thus homogeneous nucleation process has certain limitations for the low concentration solution in crystallization. In comparison to homogenous nucleation,with the presence of nucleating agents,the heterogeneous nucleation of the protein crystals faces relatively small resistance and barriers in this process.Hence the use of nucleating agents has critical significance for low-crystallizability proteins or low-initial-concentration crystallization process.With the development of the structural biology,nu-cleating agents is still a hot issue in crystallization methodology studies. The adsorption effect of porous microspheres is beneficial to the formation of disordered protein molecular clusters,and thus could promote protein nucleation.Adding porous microspheres will lead to both the increment of the number of crystallization scree- ning hits and the quality of the resultant crystals.The use of artificial seed crystal could make protein molecules orderly arranged,and keep a low-concentration condition throughout the crystal nucleation growth process.And the crosslinked crystal seed has greater ap-plication potential due to its higher stability.The novel cross-diffusion microbatch plate enables the crystallization system to realize concentration adjusting through a relatively slow cross-diffusion process and to finally reach mutual equilibrium,and in consequence, causes significant increment in the protein crystalline screening hits and the crystal quality improvement,as the number of crystalliza-tion screening hits for protein protease K increases from 39 to 47,and the diffraction resolution of crystal increases from 1.66 ?to 1.54 ?.Some properties of the substrate,such as electrostatic effect,hydrophobic interaction and hydrogen bonding,can be also used to promote the protein molecules aggregation and nucleation. This paper summarizes the influence which nucleating agents exert on the protein crystallization from two perspectives of physi-cal and chemical interactions,also roughly discusses the future prospect and research directions.%获取蛋白质晶体是蛋白质三维结构解析、医疗药品生产、自组装纳米体系构建等过程中重要的步骤.例如,利用 X射线衍射技术对蛋白质进行三维结构解析时,首先需要通过结晶条件筛选,获得质量较高的蛋白质晶体,进而进行衍射得到蛋白质结构相关信息.蛋白质结晶需要经历从未饱和区经亚稳区至形核区的形核过程以及从形核区到亚稳区的生长成熟过程.在整个蛋白质结晶过程中,形核过程是至关重要的一步.均相形核过程中,结晶体系中各个部分形核概率相同,当蛋白质结晶体系中溶液的过饱和度足够克服形核势垒时,在形核区发生成核,因而在低浓度的结晶溶液体系中,均相形核存在一定的局限性.形核剂的添加使蛋白质晶体异相形核,相较于均相形核其需要克服的阻力小,形核势垒低.因而形核剂的使用对于难结晶蛋白或者起始浓度过低的蛋白质结晶具有重要意义.随着结构生物学的发展,形核剂在蛋白质结晶中的研究仍是结晶方法学领域的热点问题.多孔微球对蛋白质分子的吸附作用有利于无序蛋白质分子团簇的形成,进而促进蛋白质形核.添加多孔微球不但可以增加结晶条件筛选数,也可以提高晶体质量.促进蛋白质分子有序排列的形核剂籽晶的使用,使晶体的形核生长过程始终处于结晶体系溶液浓度较低的状态,而交联的籽晶因为稳定性更高而更有应用前景.新型交互扩散结晶板中,蛋白质结晶体系通过一个较缓慢的交互扩散过程实现蛋白质结晶溶液浓度的变化,并且结晶体系可达到共平衡,因而能显著提高蛋白质晶体结晶条件筛选数和晶体质量:蛋白酶K结晶条件数由 39 个提升至47 个,分辨率由 1.66 ?提升至 1.54 ?.利用基底材料的一些特性,如静电作用、疏水作用和氢键,可以起到促进蛋白质分子聚集的功能,从而促进形核.本文从物理作用和化学作用两个角度详细总结了形核剂对蛋白质结晶的影响,并展望了该领域的发展前景及研究方向.
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