Clathrin is a ~630 kDa protein that resembles a three-legged pinwheel, with each leg having a height of ~50 nm. Inside cells, clathrin self-assembles with the help of adaptor proteins to form cage-like structures that facilitate the encapsulation of extracellular material by the cell membrane and transportation into the cell cytoplasm. In vitro, clathrin can self-assemble without the need for lipid membranes or adaptor proteins to form a variety of three-dimensional shapes like tetrahedra, cubes, and spheres as well as two-dimensional sheets. The underlying mechanisms that enable clathrin to self-assemble into such a diversity of nanostructures is not well understood. Here we utilize Cryo Transmission Electron Microscopy (cryo-TEM) and Dynamic Light Scattering (DLS) to explore the influence of pH and ionic concentration on clathrin self-assembly and to discover potential new nanostructures that clathrin may be able to form.Our results demonstrate that clathrin has different assembly properties in 100 mM MES at pH 5 versus pH 6. Within 30 minutes of inducing self assembly, large fused aggregates ~200-600 nm were found at pH 5; in contrast, in pH 6, spherical cages of diameter ~60-100 nm were observed under cryo-TEM. Interestingly, upon aging for 4 days at room temperature, the large aggregates initially observed at pH 5 remodeled to form 60-100 nm cages, while the sample assembled at pH 6 remained unchanged. This corroborates DLS data that indicate clathrin self-assembly is a function of both pH and time. The stability of clathrin cages at pH 6 and transformation/evolution of cages at pH 5 lend new insight into the role of kosmotropic and chaotropic ionic interactions in mediating protein self-assembly via electrostatic interactions. Current efforts are underway to construct a phase diagram of clathrin nanostructures and to compare our experimental results with computational predictions.

Self-assembly of Clathrin protein nanostructures

243rd National Meeting (2012)

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