Membrane Binding Kinetics of Peripheral Proteins Studied with Single Giant Unilamellar Vesicles

Abstract

Clathrin mediated endocytosis (CME) requires coordinated recruitment of peripheral proteins such as epsin, endophilin and amphiphysin. Recently, lots of efforts have been focused on mapping out the temporal and spatial organization of peripheral proteins during CME. However, the molecular basis defining the behavior of each protein is still not clear. In this work, we studied the association and dissociation kinetics of the membrane binding epsin N-terminal homology (ENTH) domain, as well as NBAR domains of endophilin and amphiphysin, with the goal to understand their membrane binding mechanisms in vitro. The kinetics measurements are performed on a single giant unilamellar vesicle (GUV) combined with micropipette aspiration. This technique allows us to strictly confine the protein association measurements to the pseudo - first order regime while a rapid dilution could be induced to allow protein dissociation measurements. The kinetic behavior of ENTH and NBAR domains was characteristically different. ENTH binds to membranes in a simple one-step manner with binding rates in agreement with surface plasmon resonance measurements. Association curves of NBAR, however, display a protein concentration dependent sigmoidal shape. In addition, the dissociation measurements of NBAR yield a linear relation between decay time and initial protein density on the GUV. Both observations indicate additional cooperativity between NBAR domains on the membrane. Furthermore, by studying the binding kinetics of BAR domain lacking N-terminal helix (H0), the protein cooperativity is found to be directly related to H0. Overall, this kinetics study yields insight into the similarities and differences in membrane binding mechanisms of CME related proteins.