Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization.
Use of glycerol, polyols and other protein structure stabilizing agents in protein crystallization.
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DOI：
10.1107/s0907444994014009
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发表时间：
1995-05
期刊：
影响因子：
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通讯作者：
Rui J Sousa
中科院分区：
文献类型：
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作者：
Rui J Sousa
用于结晶的蛋白质制剂应该是均匀的，并且应该在整个延长的结晶实验过程中保持均匀。制备纯蛋白质和防止其共价修饰（通过蛋白水解、巯基氧化等）的一般方法在长时间孵育中的作用是众所周知的。晶体学家不太了解用于稳定蛋白质以防止非共价修饰（部分变性、异质聚集）的一般方法，所述非共价修饰也可将结构异质性引入蛋白质制剂中。与这个问题相关的是抑制蛋白质构象柔性的方法，构象柔性可以是动态结构异质性的来源，并且呈现出对结晶的熵障碍。然而，多年来，生物化学文献中已经描述了稳定蛋白质结构的试剂。最近最广泛使用的这些结构稳定剂，甘油，用于结晶T7 RNA聚合酶。观察到这种化合物具有一般的结构稳定作用，并且它对至少这一种蛋白质的结晶是必不可少的，这导致了这样的建议，即它通常可能用于使柔性蛋白质结晶，并在结晶蛋白质的无序片段中诱导有序。随后，甘油用于许多蛋白质的结晶，效果良好。最近的其他结果表明，将晶体浸泡在含有甘油的溶液中可以对晶体蛋白质产生“结构排序”作用。这些观察结果支持了甘油在蛋白质结晶中的用途，并表明生化文献中有关蛋白质结构稳定剂的信息将在蛋白质晶体生长领域找到有用的应用。
A protein preparation to be used for crystallization should be homogeneous and should remain so throughout the course of a prolonged crystallization experiment. General methods for preparation of pure proteins and for prevention of their covalent modification (through proteolysis, sulfhydryl oxidation, etc.) during prolonged incubation are well known. Crystallographers are less aware of general methods for stabilization of proteins against non-covalent modifications (partial denaturation, heterogeneous aggregation) which can also introduce structural heterogeneity into a protein preparation. Related to this issue are methods to suppress protein conformational flexibility which can be a source of dynamic structural heterogeneity and which presents an entropic barrier to crystallization. However, for many years agents which stabilize protein structure have been described in the biochemical literature. Recently the most widely used of these structure-stabilizing agents, glycerol, was used to crystallize T7 RNA polymerase. The observation that this compound has general structure-stabilizing effects and that it was essential for crystallization of at least this one protein led to the suggestion that it might be generally useful in crystallizing flexible proteins and in inducing order in disordered segments of crystalline proteins. Subsequently, glycerol was used with good effect in the crystallization of a number of proteins. Other recent results suggest that soaking crystals in solutions containing glycerol can have 'structure-ordering' effects on the crystalline protein. These observations support the utility of glycerol in protein crystallization and suggest that the information in the biochemical literature on protein structure-stabilizing agents will find useful application in the field of protein crystal growth.