Developing a Fluorescence-based Approach to Screening for Macromolecule Crystallization Conditions.
Developing a Fluorescence-based Approach to Screening for Macromolecule Crystallization Conditions.
复制标题
开发基于荧光的方法来筛选大分子结晶条件。
DOI：
10.1021/cg1013522
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发表时间：
2011
影响因子：
3.8
通讯作者：
Pusey,MarcL
中科院分区：
文献类型：
--
作者：
Pusey,MarcL
目前的大分子结晶筛选方法依赖于随机测试结晶条件，希望一个或多个结晶将产生积极的结果。大多数平板的结果要么是澄清的，要么是沉淀的溶液，其中的结果通常被实验者丢弃。然而，这些中的许多实际上可能接近结晶条件，这一事实被明显结果的性质所掩盖。我们正在开发一种基于荧光的方法来确定结晶条件，这种方法也可用于评估可能接近于产生晶体的条件。该方法使用荧光各向异性和强度的测量。首先使用模型蛋白测试该方法，通过荧光测量确定可能的结果，其中板数据显示澄清或沉淀的溶液进行优化筛选。结果表明，结晶条件的数量增加了83%。然后尝试将该方法作为多种测试蛋白质的唯一筛选方法。在每种情况下，至少发现了一种或多种结晶条件，据估计，其中约53%的结晶条件不会使用平板筛发现。
Current macromolecule crystallization screening methods rely on the random testing of crystallization conditions, in the hope that one or more will yield positive results, crystals. Most plate outcomes are either clear or precipitated solutions, in which the results are routinely discarded by the experimenter. However, many of these may in fact be close to crystallization conditions, a fact which is obscured by the nature of the apparent outcome. We are developing a fluorescence-based approach to the determination of crystallization conditions, an approach which can also be used to assess conditions that may be close to those that would give crystals. The method uses measurements of fluorescence anisotropy and intensity. The method was first tested using model proteins, with likely outcomes as determined by fluorescence measurements where the plate data showed either clear or precipitated solutions being subjected to optimization screening. The results showed a ∼83% increase in the number of crystallization conditions. The method was then tried as the sole screening method with a number of test proteins. In every case, at least one or more crystallization conditions were found, and it is estimated that ∼53% of these would not have been found using a plate screen.