Structure function analysis of thioredoxin from Wuchereria bancrofti, a drug target for human lymphatic filariasis Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften an der Fakultät für Mathematik, Informatik und Naturwissenschaften der Universität Hamburg vorgelegt von Nasser Yousef (M.Sc.) aus Kairo, Ägypten Hamburg 2014 Die vorliegende Arbeit wurde im Zeitraum von 2009 bis 2014 in der Arbeitsgruppe von Prof. Ch. Betzel am Institut für Biochemie und Molekularbiologie am Department Chemie der Universität Hamburg angefertigt. Gutachter: Herr Prof. Christian Betzel Herr Prof. Reinhard Bredehorst Tag der Disputation: 25.07.2014 To my wife and daughters Table of Contents Table of Contents Table of Contents ........................................................................................................................I List of figures ........................................................................................................................... VI List of tables ............................................................................................................................. IX List of abbreviations ................................................................................................................. X Symbols for Amino Acids .................................................................................................... XVI 1. Aim of this Work .................................................................................................................. 1 2. Summary – Zusammenfassung ........................................................................................... 3 2.1 Summary .............................................................................................................................. 3 2.2 Zusammenfassungen ............................................................................................................ 5 3. Introduction .......................................................................................................................... 8 3.1 Lymphatic filariasis .......................................................................................................... 8 3.1.1 Biology and epidemiology ......................................................................................... 8 3.1.2 Transmission and infection ........................................................................................ 9 3.1.3 Identification methods for W. bancrofti .................................................................. 11 3.1.4 Pathology of lymphatic filariasis ............................................................................. 13 3.1.5 Diagnosis and treatment of lymphatic filariasis ...................................................... 15 3.2 Thioredoxin .................................................................................................................... 18 3.2.1 Biochemistry of thioredoxins .................................................................................. 18 3.2.2 Structure of thioredoxins ......................................................................................... 21 3.2.3 Conserved residues in thioredoxins ......................................................................... 23 3.2.4 Reaction mechanism of thioredoxins ...................................................................... 25 3.2.5 Thermodynamic stability of Trx .............................................................................. 27 3.2.6 Trapping of Trx substrate ........................................................................................ 27 3.2.7 Diversity within an ubiquitous family ..................................................................... 30 I Table of Contents 3.2.7.1 Bacterial thioredoxin ......................................................................................... 30 3.2.7.2 Yeast and mammalian thioredoxins .................................................................. 31 3.2.7.3 Plant thioredoxins ............................................................................................. 32 4. Materials and Methods ...................................................................................................... 33 4.1 Materials ......................................................................................................................... 33 4.1.1. Plasmid ................................................................................................................... 33 4.1.2. Laboratory equipments ........................................................................................... 34 4.1.5. Buffers and solutions .............................................................................................. 35 4.1.6 Bacteria culture media ............................................................................................. 36 4.2 Methods .......................................................................................................................... 38 4.2.1 Molecular biology methods ..................................................................................... 38 4.2.1.1 Polymerase chain reaction (PCR) ..................................................................... 38 4.2.1.2 Cloning of WbTrx-1 in pRSET B vector without His-tag ................................ 38 4.2.1.3 Purification of DNA .......................................................................................... 39 4.2.1.4 Restriction digestion of DNA fragments .......................................................... 39 4.2.1.5 Dephosphorylation of plasmid DNA ................................................................ 39 4.2.1.6 Ligation ............................................................................................................. 39 4.2.2 Mutagenesis ............................................................................................................. 40 4.2.2.1 Site directed mutagenesis .................................................................................. 40 4.2.2.2 Site directed mutagenesis of overlap extension ................................................ 41 4.2.2.3 Removal of template-DNA from a PCR reaction ............................................. 41 4.2.2.4 DNA purification .............................................................................................. 41 4.2.2.5 Agrose gelelectrophoresis ................................................................................. 41 4.2.3 Preparation of chemically competent cells with CaCl2 ........................................... 42 4.2.4 Isolation and purification of plasmids ..................................................................... 42 II Table of Contents 4.2.5 DNA-Sequencing..................................................................................................... 42 4.2.6 Transformation of plasmid into E.coli cells ............................................................ 42 4.2.7 Preparation of glycerol stocks ................................................................................. 43 4.2.8 Cell disruption for protein purification .................................................................... 43 4.2.9 Regeneration of Q-sepharose column ...................................................................... 43 4.2.10 Expression of WbTrx-1 wild type and WbTrx-1-C39S variant ............................. 44 4.2.11 Purification of WbTrx-1 wild type and WbTrx-1-C39S variant ............................ 44 4.2.11.1 Ion exchange chromatography ............................................................................ 44 4.2.11.2 Fast Protein Liquid Chromatography (FPLC) ................................................ 44 4.2.12 Protein quantification with Nanodrop 2000c......................................................... 45 4.2.13 Enzymatic Assay for thioredoxin (WbTrx-1) ........................................................ 45 4.2.14 Inhibition of thioredoxin (WbTrx-1)...................................................................... 45 4.2.15 SDS-polyacrylamide gel electrophoresis (PAGE) ................................................ 46 4.2.16 Crystallization experiments ................................................................................... 46 4.2.16.1 Crystallization of WbTrx-1 and WbTrx-1-C39S ............................................. 46 4.2.16.2 Dynamic Light Scattering (DLS) .................................................................... 47 4.2.16.3 Pre-Crystallization Test .................................................................................. 47 4.2.16.4 Robotic Screening ........................................................................................... 48 4.2.16.6 Optimization of WbTrx-1-C39S Crystals ....................................................... 49 4.2.16.7 Data Collection ............................................................................................... 49 4.2.16.8 Matthews Coefficient (VM) ............................................................................. 49 4.2.16.9 Cryogenic Techniques .................................................................................... 49 4.2.16.10 Macromolecular Crystallography Beamline X13 ......................................... 50 4.2.17 Model Building and Refinement ........................................................................... 50 4.2.17.1 Coot: Crystallographic Object-Oriented Toolkit ...........................................