title: Kinetics of endophilin N-BAR domain dimerization and membrane interactions.
abstract: The recruitment to plasma membrane invaginations of the protein endophilin is a temporally regulated step in clathrin-mediated endocytosis. Endophilin is believed to sense or stabilize membrane curvature, which in turn likely depends on the dimeric structure of the protein. The dynamic nature of the membrane association and dimerization of endophilin is thus functionally important and is illuminated herein. Using subunit exchange Förster resonance energy transfer (FRET), we determine dimer dissociation kinetics and find a dimerization equilibrium constant orders of magnitude lower than previously published values. We characterize N-BAR domain membrane association kinetics under conditions where the dimeric species predominates, by stopped flow, observing prominent electrostatic sensitivity of membrane interaction kinetics. Relative to membrane binding, we find that protein monomer/dimer species equilibrate with far slower kinetics. Complementary optical microscopy studies reveal strikingly slow membrane dissociation and an increase of dissociation rate constant for a construct lacking the amphipathic segment helix 0 (H0). We attribute the slow dissociation kinetics to higher-order protein oligomerization on the membrane. We incorporate our findings into a kinetic scheme for endophilin N-BAR membrane binding and find a significant separation of time scales for endophilin membrane binding and subsequent oligomerization. This separation may facilitate the regulation of membrane trafficking phenomena.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Capraro
authors.forename: Zheng
authors.initials: Z
authors.lastname: Shi
authors.forename: Tingting
authors.initials: T
authors.lastname: Wu
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Chen
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Dunn
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Rhoades
authors.forename: Tobias
authors.initials: T
authors.lastname: Baumgart
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008567:Membranes, Artificial; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D008567:Membranes, Artificial; D000217:Acyltransferases; C018764:2-acylglycerophosphate acyltransferase
keywords: Endocytosis; Kinetics; Membrane Biophysics; Membrane Curvature; Membrane Trafficking; Protein Dimerization; Protein Self-assembly; Protein-Membrane Interactions