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Investigations of the Mechanism for Activation of Bacillus Thuringiensis Phosphatidylinositol-Specific Phospholipase C

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Investigations of the Mechanism for Activation of Bacillus Thuringiensis Phosphatidylinositol-Specific Phospholipase C Investigations of the Mechanism for Activation of Bacillus Thuringiensis Phosphatidylinositol-specific Phospholipase C Author: Mingming Pu Persistent link: http://hdl.handle.net/2345/1179 This work is posted on eScholarship@BC, Boston College University Libraries. Boston College Electronic Thesis or Dissertation, 2009 Copyright is held by the author, with all rights reserved, unless otherwise noted. Boston College The Graduate School of Arts and Sciences Department of Chemistry INVESTIGATIONS OF THE MECHANISM FOR ACTIVATION OF BACILLUS THURINGIENSIS PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C a dissertation by MINGMING PU submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy August 2009 © copyright by MINGMING PU 2009 Investigations of the Mechanism for Activation of Bacillus thuringiensis Phosphatidylinositol-specific Phospholipase C Mingming Pu Under the direction of Dr. Mary F. Roberts ABSTRACT The bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is specifically activated by low concentrations of a non-substrate lipid, phosphatidylcholine (PC), presented as an interface. However, if the PC concentration in the interface is too high relative to substrate, the enzyme exhibits surface dilution inhibition. Understanding this bacterial enzyme, which shares many kinetic features with the larger and more complex mammalian PI-PLC enzymes, requires elucidating the mechanism for PC activation and inhibition. Various techniques were applied to study the interaction of the protein with vesicles composed of both the activator lipid PC and the substrate lipid (or a nonhydrolyzable analogue). Fluorescence correlation spectroscopy (FCS), used to monitor bulk partitioning of the enzyme on vesicles, revealed that both the PC and the substrate analogue are required for the tightest binding of the PI-PLC to vesicles. Furthermore, the tightest binding occurred at low mole fractions of substrate- like phospholipids. Field cycling 31P NMR (fc-P-NMR) spin-lattice relaxation studies provided information on how bound protein affects the lipid dynamics in mixed substrate analogue/PC vesicles. The combination of the two techniques could explain the enzyme kinetic profile for the PC activation and surface dilution inhibition: small amounts of PC in an interface enhanced PI-PLC binding to substrate-rich vesicles while high fractions of PC tended to sequester the enzyme from the bulk of its substrate leading to reduced specific activity. FCS binding profiles of mutant proteins were particularly useful in determining if a specific mutation affected a single or both phospholipid binding modes. In addition, an allosteric PC binding site was identified by fc-P-NMR and site directed spin labeling. A proposed model for PC activation suggested surface-induced dimerization of the protein. Experiments in support of the model used cysteine mutations to create covalent dimers of this PI-PLC. Two of these disulfide linked dimers, formed from W242C or S250C, exhibited higher specific activities and tighter binding to PC surfaces. In addition, single molecule total internal reflection fluorescence microscopy was used to monitor the off-rate of PI-PLC from surface tethered vesicles, providing us with a direct measure of off-rates of the protein from different composition vesicles. Acknowledgements First of all I would like to especially thank my advisor Prof. Mary F. Roberts for her education, support, and help during my Ph.D. studies. She’s always encouraging and supportive, giving me confidence and has inspired me in the research. I am so lucky to have her as my advisor and her great personality will influence me for the rest of my life. I would like to thank all the collaborators, Prof. Anne Gershenson for her guidence in the FCS and single molecule projects; Prof. Thomas Chiles for his guidance in the U937 cell viability studies; and Prof. Jeff Gelles for the TIR-FM studies. The collaboration experience is especially valuable to me. Special thanks are expressed to Dr. Larry Friedman, Dr. Derek Blair and Dr. Xiaomin Fang. These nice and smart people helped me a lot when I start these projects. I would like to thank all members of the Prof. Roberts group, the present and the past, for their friendship and helpful discussions. I shared a lot of fun time with Xiaomeng Shi, Yang Wei and Su Guo. Xin Zhang has helped me in learing new techniques during my first year. Dr. Wei Chen, and Dr. Yanling Wang have provided me with useful suggestions. I have enjoyed graduate study largly because of thse people around me. Lastly and most importantly, I want to thank my parents for everything they have done for me, for their love and care during my whole life. I want to thank my husband, Weixing, for his love and support, and for the happiness he brings to my life. Table of Contents List of Abbreviations .......................................................................................................... v List of Figures .................................................................................................................. viii List of Tables ..................................................................................................................... xi Chapter 1 Introduction ................................................................................................... 1 1.1 Phospholipids ....................................................................................................... 1 1.1.1 Membrane lipids ........................................................................................... 1 1.1.2 Structure of phospholipids ............................................................................ 1 1.1.3 Phospholipid aggregation.............................................................................. 2 1.2 Phospholipases ..................................................................................................... 5 1.2.1 Peripheral enzymes and their biological functions ....................................... 5 1.2.2 Phospholipases .............................................................................................. 7 1.2.3 Phosphatidylinositol-specific Phospholipase C ............................................ 9 1.3 Interfacial catalysis ............................................................................................. 18 1.4 PC activation of PI-PLC ..................................................................................... 23 1.5 Membrane binding assays for peripheral proteins ............................................. 25 1.5.1 Membrane binding assays ........................................................................... 25 1.5.2 Kinetics of membrane-protein interactions ................................................. 28 1.6 Thesis directions ................................................................................................. 28 Chapter 2 Materials and Methods ................................................................................ 31 2.1 Chemicals ........................................................................................................... 31 2.2 Lipids .................................................................................................................. 32 2.2.1 Enzymatic synthesis of dimyristoyl-d54-phosphatidylmethanol (d54- DMPMe) ................................................................................................................... 32 2.2.2 Vesicle Preparation and Sizing by Dynamic Light Scattering (DLS) ........ 33 2.3 Enzymatic synthesis and purification of cIP ...................................................... 35 2.4 B. thuringiensis PI-PLC expression and purification ......................................... 37 2.4.1 Over-expression in Escherichia coli cells with a plasmid containing the gene for B. thuringiensis PI-PLC .............................................................................. 37 2.4.2 Purification of PI-PLC ................................................................................ 37 2.5 Construction of PI-PLC mutants ........................................................................ 39 i 2.6 Modification of PI-PLC ..................................................................................... 41 2.6.1 Covalent dimer formation ........................................................................... 41 2.6.2 PI-PLC site-directed spin labeling .............................................................. 41 2.6.3 PI-PLC specific Cys labeling with Alexa Fluor 488 .................................. 42 2.7 Binding of PI-PLC to SUVs by centrifugation-filtration assay ......................... 43 2.8 31P and 1H NMR at 11.7 T .................................................................................. 44 2.8.1 Enzyme kinetic assay by 31P NMR ............................................................. 44 2.8.2 Binding study of spin labeled PI-PLC to SUVs by 31P and 1H NMR......... 47 2.9 High resolution 31P field cycling NMR .............................................................. 51 2.10 Fluorescence Correlation Spectroscopy study of Alexa Fluor 488 labeled PI- PLC binding to SUVs ................................................................................................... 56 2.11 Single Molecule Total Internal Reflection Fluorescence Microscopy (TIR-FM) study of Alexa488 labeled PI-PLC binding to surface tethered SUVs ......................... 62 2.11.1 Surface
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