FBS Colloquia No.407Laboratory for Supramolecular Crystallography

Protein structures provide valuable information for understanding their molecular functions. Among the methods to determine protein structures, X-ray crystallography allows for rapid structure determination due to advances in synchrotron facilities and analysis software. However, obtaining crystals remains a bottleneck in X-ray crystallography. Even when crystals are obtained, issues such as low resolution often arise. For GPCRs, which are seven-transmembrane proteins, issues like these have been addressed by using fusion proteins such as T4 lysozyme or thermostabilized apocytochrome b562 (E. Chun et al., Structure 20, 967 (2012)). Our laboratory has established a method for crystallizing membrane proteins using a fusion protein consisting of a nanobody against the fluorescent protein GFP, employing the lipid cubic phase method (Japanese Patent No. 7657406). In this method, because a complex has the fluorescent protein, it is easier to determine whether a crystal is indeed a protein crystal, allowing diffraction experiments to proceed. Additionally, the formation of a GFP-Nanobody complex increases the hydrophilic regions, which enhances protein-protein interactions within crystals and can be expected to acquire high-quality crystals. I would also like to discuss the practical application of this approach to extracellular proteins, which present difficulties in preparing homogeneous samples because of glycosylation.