Nucleotide-Dependent Dimerization and Conformational Switching of Atlastin.

A common feature of dynamin-related proteins (DRPs) is their use of guanosine triphosphate (GTP) to control protein dynamics. In the case of the endoplasmic- reticulum- (ER)-resident membrane protein atlastin (ATL), GTP binding and hydrolysis result in membrane fusion of ER tubules and the generation of a branched ER network. In this chapter, we describe two independent methods for dissecting the mechanism underlying nucleotide-dependent quaternary structure and conformational changes of ATL, focusing on size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and Förster resonance energy transfer (FRET), respectively. The high temporal resolution of the FRET-based assays enables the ordering of the molecular events identified in structural and equilibrium-based SEC-MALS studies. In combination, these complementary methods report on the oligomeric states of a system at equilibrium and timing of key steps along the enzyme's catalytic cycle. These methods are broadly applicable to proteins that undergo ligand-induced dimerization and/or conformational changes.