Characterization of protein samples using dynamic light scattering

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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.