Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2014 Investigation of SNARE-mediated exocytosis controlled by synaptic regulatory proteins Jaeil Shin Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Biochemistry Commons Recommended Citation Shin, Jaeil, "Investigation of SNARE-mediated exocytosis controlled by synaptic regulatory proteins" (2014). Graduate Theses and Dissertations. 13995. https://lib.dr.iastate.edu/etd/13995 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Investigation of SNARE-mediated exocytosis controlled by synaptic regulatory proteins by Jaeil Shin A dissertation submitted to graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Biochemstry Program of Study Committee: Yeon-Kyun Shin, Major Professor Guru Rao Scott Nelson Michael Shogren-Knaak Edward Yu Iowa State University Ames, Iowa 2014 Copyright © Jaeil Shin, 2014. All rights reserved. ii TABLE OF CONTENTS ABSTRACT ................................................................................................................................ vi CHAPTER 1: GENERAL INTRODUCTION ...........................................................................1 1.1 Membrane fusion and exocytosis ..........................................................................................1 1.2 SNAREs ................................................................................................................................1 1.3 SNARE-mediated neuroexocytosis .......................................................................................3 1.4 Regulation of neurotransmitter release .................................................................................4 1.4.1 Synaptotagmin-1..............................................................................................................4 1.4.2 Sec1/Munc18-1 (SM) protein family ..............................................................................5 1.4.3 Complexin-1 .............................................................................................................................. 6 1.5 Investigation apparatus ..........................................................................................................7 1.5.1 Nanodisc as a model membrane: reconstitution of a single membrane protein .............7 1.5.2 Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) ..........8 1.5.3 Single molecular fluorescence (Förster) resonance energy transfer (smFRET) ............9 1.6 References ...........................................................................................................................11 1.7 Figures and Legends ............................................................................................................19 CHAPTER 2: MULTIPLE CONFORMATIONS OF A SINGLE SNAREPIN BETWEEN TWO NANODISC MEMBRANES REVEAL DIVERSE PRE-FUSION STATES .......................................................................................................................................34 2.1 Abstract ...............................................................................................................................34 2.2 Introduction .........................................................................................................................35 2.3 Results .................................................................................................................................36 2.3.1 Reconstitution of the SNAREpin into two nanodiscs ..................................................36 iii 2.3.2 The t- and v-SNAREs assemble into a parallel configuration between two nanodiscs, some with the frayed C-terminal end ...................................................................37 2.3.3 EPR confirms that both the fully assembled four-helix bundle and the half-zipped complex represent trans-SNAREpin conformations .............................................................38 2.3.4 The real-time analysis of single SNAREpins reveals a subpopulation with highly fluctuating dynamic ...............................................................................................................40 2.4 Discussion ...........................................................................................................................41 2.5 Materials and Methods ........................................................................................................43 2.5.1 Protein expression, purification, and fluorophore- or spin-labeling .............................43 2.5.2 Reconstitution and purification of SNARE-incorporated nanodiscs ............................45 2.5.3 Lipid mixing between v-discs and either t-discs or t-liposomes ...................................46 2.5.4 FRET measurements of trans-SNARE complex with TIR ..........................................46 2.5.5 Purification and measurement of spin labeled trans-SNARE complex in EPR spectroscopy ..........................................................................................................................47 2.6 References ...........................................................................................................................49 2.7 Figures and Legends ............................................................................................................52 2.8 Supporting Information .......................................................................................................58 CHAPTER 3: SYNAPTOTAGMIN 1 IS AN ANTAGONIST FOR MUNC18-1 IN SNARE-ZIPPERING .................................................................................................................65 3.1 Abstract ...............................................................................................................................65 3.2 Introduction .........................................................................................................................66 3.3 Results .................................................................................................................................67 3.3.1 Munc18-1 promotes SNARE zippering without syt1, but not in the presence of syt1 .67 3.3.2 Syt1 and Munc18-1 are mutually antagonistic in SNARE-dependent lipid mixing ....69 iv 3.3.3 Munc18-1 has little effect on Ca2+-triggered content mixing ......................................70 3.4 Discussion ...........................................................................................................................71 3.5 Materials and Methods ........................................................................................................73 3.5.1 Plasmid constructs and site-directed mutagenesis ........................................................73 3.5.2 Protein expression and purification ..............................................................................74 3.5.3 Lipid mixture preparation .............................................................................................74 3.5.4 Fluorophore-labeling of the single cysteine mutants ....................................................75 3.5.5 Reconstitution and purification of t- and v-discs .........................................................76 3.5.6 FRET measurements of trans-SNAREpins ..................................................................76 3.5.7 proteoliposomes reconstitution .....................................................................................77 3.5.8 Bulk lipid mixing assay ................................................................................................79 3.5.9 Single vesicle docking and lipid mixing assays ...........................................................79 3.5.10 Single-vesicle content mixing assay ...........................................................................80 3.6 References ...........................................................................................................................81 3.7 Figures and Legends ............................................................................................................86 3.8 Supporting Information .......................................................................................................90 CHAPTER 4: THE ROLE OF COMPLEXIN-1 N-TERMINUS AS AN ACTIVE ASSISTANT IN Ca2+ TRIGGERED FUSION PORE FORMATION ..................................95 4.1 Abstract ...............................................................................................................................95 4.2 Introduction .........................................................................................................................95 4.3 Results .................................................................................................................................98 4.3.1 Cpx1 facilitates content mixing events in the presence of syt1 and Ca2+ .....................98 4.3.2 SNARE motif in the trans-SNAREpin is fully assembled by cpx1 .............................98 4.3.3 Cpx1 increases vesicle-vesicle lipid mixing .................................................................99