Protein Domain Organization in the Nuclear Pore Complex Studied by Fluorescence Anisotropy

Abstract

Fluorescence anisotropy measurements can yield information about the mobility, orientation and proximity of fluorophores and can be used to study the dynamics of proteins in vivo. The nuclear pore complex (NPC) is a large macromolecular complex, the size and complexity of which present experimental challenges. The dynamics of nucleoporins (nups) were examined using fluorescence polarization microscopy. Using a theoretical framework which exploits the symmetry of the NPC and its organization in the nuclear envelope we can resolve the order and disorder of individual protein domains within the complex. Specific domains of individual nups were tagged with GFP and examined using fluorescence polarization microscopy. We characterized the domain organization of the FG nups, which are required for transport of cargo through the NPC. This approach revealed both structured and unstructured domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. This technique allows the collection of structural information in vivo with the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nups, which are implicated in the mechanism of cargo transport.