(588c) Self-Assembly of Clathrin Protein 3D Structures

Clathrin is a ~630 kDa protein that resembles a three-legged pinwheel, i.e., a triskelion, with each leg having an extended length of ~50 nm and width of ~ 3 nm. Due to molecular recognition interactions between legs and the non-planar geometry of the basic building block, clathrin self-assembles to form 3D assemblies resembling fullerene structures. In vivo, clathrin helps in transportation of extracellular components across the cell membrane with the help of adaptor proteins through receptor mediated endocytosis by forming enclosed polyhedral cages. In vitro, clathrin self-assembles in the absence of lipid membranes or adaptor proteins to form a variety of three-dimensional shapes like tetrahedra, cubes and spheres. The underlying mechanisms that enable clathrin to self-assemble into such a diversity of nanostructures is not well understood. Here we use Cryo Transmission Electron Microscopy (cryo-TEM) and Dynamic Light Scattering (DLS) to explore the influence of solvent conditions (pH and ionic strength) on the kinetics and morphology of clathrin self-assembly and to discover potential new nanostructures that clathrin may form.

Our results demonstrate that clathrin has strikingly different assembly kinetics at pH 5 (below the isoelectric point) versus pH 6 (above the isoelectric point). Within 30 minutes of inducing self-assembly at pH 6, discrete spherical cages of diameter ~60-100 nm were observed under cryo-TEM. In contrast, inducing assembly at pH 5 causes the formation of large (~200-600 nm) disordered aggregates. Interestingly, upon aging for 4 days at room temperature, the large aggregates undergo substantial remodeling to form discrete 60-100 nm spherical cages, while the samples assembled at pH 6 remained unchanged. This corroborates DLS data that indicate clathrin self-assembly is a function of both pH and time, with large length-scale structural rearrangements occuring over long time-scales (i.e., days). The stability of clathrin cages at pH 6 and the transformation of cages at pH 5 lend new insight into the role of kosmotropic and chaotropic interactions in mediating protein self-assembly via electrostatic interactions. We hypothesize that clathrin initially forms large, irregular aggregates at pH 5 due to the sudden decrease in electrostatic repulsion between building blocks that is experienced below the isoelectric point. Over time, the mechanical strain within the aggregate is resolved by reorganization of clathrin triskelion into discrete cage structures. Current efforts are underway to construct a phase diagram of clathrin nanostructures and to compare our experimental results with computational predictions that consider the strength of leg-leg interactions and the elasticity of individual legs.