Characterization of protein samples using dynamic light scattering

一般来说，解决蛋白质三维结构的瓶颈是结晶。 需要优化材料的来源、纯度以及各种物理化学参数，如温度、pH值或离子强度，以有利于结晶过程。 虽然可以使用标准蛋白质纯化技术实现样品纯度，但这些方法不能消除构象异质性。这些异质性通常是由于蛋白质的柔性，部分解折叠或蛋白质聚集，并且是蛋白质结晶中的严重问题。 蛋白质的异质性也危及使用电子显微镜研究蛋白质复合物，因为这种方法依赖于平均数以千计的图像代表相同的大分子组装。 因此，评估用于结构分析的任何样品的均匀性至关重要。 光散射代表了这种分析的最强大和最有效的技术。 虽然多角度光散射评估绝对分子量，并反过来，蛋白质复合物的化学计量，因为它们被纯化，准弹性光散射评估样品的均匀性程度。 使用这两种技术对给定样品的分析可以有效地“预测”蛋白质样品是否适合通过冷冻电子显微镜进行晶体形成或3D重建。 因此，光散射可以在几分钟内用非常少量的样品确定是否值得对给定的复合物进行进一步的结构分析。 由于蛋白质结晶是一个需要数月的过程，需要毫克的高度纯化的蛋白质，这种类型的分析的重要性不能过分强调。 事实上，光散射系统在任何晶体学实验室都是必须的，因此，经过这种培训的HQP有望掌握这种方法。 三个实验室（两个晶体学实验室和一个电子显微镜实验室）之间共享该设备进一步最大限度地利用了资源，同时极大地增强了与生物学和医学相关的相关领域的研究项目。

Generally the bottleneck in solving the three-dimensional structure of a protein is crystallization. The source of the material, its purity as well as a variety of physical-chemical parameters such as temperature, pH or ionic strength need to be optimized in order to favor the crystallization process. While sample purity can be achieved using standard protein purification techniques, these methods do not eliminate conformational heterogeneity. These heterogeneities are usually due to protein flexibility, partial unfolding or protein aggregation and are a severe problem in protein crystallization. Protein heterogeneities also jeopardize the study of protein complexes using electron microscopy since this method relies on averaging thousand of images representing identical macromolecular assemblies. Therefore, it is vital to assess the homogeneity of any sample intended for structural analysis. Light scattering represents the most powerful and efficient technique for such analysis. While multi-angle light scattering assesses absolute molecular weights and, in turn, stoichiometries of protein complexes as they are purified, quasi-elastic light scattering evaluates the degree of sample homogeneity. The analysis of a given sample using these two techniques can effectively 'forecast' if a protein sample is suitable for crystal formation or 3D reconstruction by cryo-electron microscopy. Therefore, light scattering can determine in minutes and with very small amounts of sample whether it is worth pursuing further structural analysis on a given complex or not. Since protein crystallization is a process that takes months and requires milligrams of highly purified protein, the importance of this type of analysis cannot be over emphasized. In fact, a light scattering system is a must in any crystallography laboratory and hence HQP with this type of training are expected to master this methodology. Sharing this equipment between three laboratories (two crystallography and one electron microscopy) further maximizes the use of resources and at the same time greatly enhances research programs in related areas that are biologically and medically relevant.